Purposes: To use optical coherence tomography (OCT) to image and quantify the effect of various corneal conditions, in terms of corneal, stromal and epithelial thickness, and light backscatter. To assess the changes caused by overnight orthokeratology (Corneal Refractive Therapy; CRT^TM) lens wear, keratoconus and laser in-situ keratomileusis (LASIK) refractive surgery, each of which may lead to topographical alterations in corneal thickness either by temporary moulding, degeneration, or permanent laser ablation, respectively. <br /><br /> Methods: Topographical thickness of the cornea was measured using OCT in all studies. The CRT^TM studies investigated myopic and hyperopic treatment, throughout the day. The myopic studies followed lens wear over a 4 week period, which was extended to 12 months, and investigated the thickness changes produced by two lenses of different oxygen transmissibility. CRT^TM for hyperopia (CRTH^TM) was evaluated after a single night of lens wear. <br /><br /> In the investigation of keratoconus, OCT corneal thickness values were compared to those obtained from Orbscan II (ORB) and ultrasound pachymetry (UP). A new fixation device was constructed to aid in the measurement of topographical corneal and epithelial thickness along 8 directions of gaze. Pachymetry maps were produced for the normal non-lens wearing cornea, and compared with the rigid gas permeable (RGP) lens wearing cornea and the keratoconic cornea. <br /><br /> Thickness changes prior to, and following LASIK were measured and monitored throughout six months. Myopic and hyperopic correction was investigated individually, as the laser ablation profiles differ for each type of procedure. The LASIK flap interface was also evaluated by using light backscatter data to monitor healing. <br /><br /> Results: Following immediate lens removal after myopic CRT^TM, the central cornea swelled less than the periphery, with corneal swelling recovering to baseline levels within 3 hours. The central epithelium decreased and mid-peripheral epithelium increased in thickness, with a more gradual recovery throughout the day. There also seemed to be an adaptation effect on the cornea and epithelium, showing a reduced amount of change by the end of the 4 week study period. The thickness changes did not alter dramatically during the 12 month extended study. In comparing the two lens materials used for myopic CRT^TM (Dk/t 91 vs. 47), there were differences in stromal swelling, but no differences in the central epithelial thinning caused by lens wear. There was a statistically insignificant asymmetry in mid-peripheral epithelial thickening between eyes, with the lens of lower Dk causing the greater amount of thickening. Hyperopic CRT^TM produced a greater increase in central stromal and central epithelial thickness than the mid-periphery. Once again, the stroma recovered faster than the epithelium, which remained significantly thicker centrally for at least six hours following lens removal. <br /><br /> Global pachymetry measurements of the normal cornea and epithelium found the periphery to be thicker than the centre. The superior cornea and epithelium was thicker than the inferior. In the measurement of the keratoconic cornea, OCT and ORB correlated well in corneal thickness values. UP measured greater values of corneal thickness. The keratoconic epithelium was thinner than normal, and more so over the apex of the cone than at the centre. The location of the cone was most commonly found in the inferior temporal region. Central epithelial thickness was thinner in keratoconics than in RGP lens wearers, which in turn was thinner than in non-lens wearers. <br /><br /> Following LASIK surgery for both myopia and hyperopia, the topographical OCT thickness profiles showed stromal thinning in the areas of ablation. The central myopic cornea showed slight regression at 6 months. During early recovery, epithelial thickness increased centrally in hyperopes and mid-peripherally in myopes. By the end of the 6 month study, mid-peripheral epithelial thickness was greater than the centre in both groups of subjects. The light backscatter profiles after LASIK showed a greater increase in backscatter on the anterior side of the flap interface (nearer the epithelium), than the posterior side (in the mid-stroma) during healing. The flap interface was difficult to locate in the OCT images at 6 months. <br /><br /> Conclusion: All the CRT^TM lenses used in this project produced more corneal swelling than that seen normally overnight without lens wear. In order for these lenses to be worn safely for long periods of time without affecting the health of the cornea, they need to be manufactured from the highest oxygen transmissible material available. The long-term effect of thinning on the epithelium's barrier properties needs to be monitored closely. <br /><br /> Global topographical thickness of the cornea and epithelium was measured using OCT in normal, RGP lens wearing and keratoconic eyes. Corneal and epithelial thickness was not symmetrical across meridians. The epithelium of RGP lens wearers was slightly thinner than normal, but not as thin as in keratoconics, suggesting that the epithelial change seen in keratoconus is mainly due to the condition. <br /><br /> Post-LASIK corneal and epithelial thickness profiles were not the same for myopic and hyperopic subjects, since the ablation patterns vary. Epithelial thickening in the mid-periphery had not recovered by six months in myopes or hyperopes, possibly indicating epithelial hyperplasia. Light backscatter profiles were used to monitor the recovery of the LASIK flap interface, showing the band of light backscatter around the flap interface to decrease as the cornea healed.

Purposes: To use optical coherence tomography (OCT) to image and quantify the effect of various corneal conditions, in terms of corneal, stromal and epithelial thickness, and light backscatter. To assess the changes caused by overnight orthokeratology (Corneal Refractive Therapy; CRT^TM) lens wear, keratoconus and laser in-situ keratomileusis (LASIK) refractive surgery, each of which may lead to topographical alterations in corneal thickness either by temporary moulding, degeneration, or permanent laser ablation, respectively. <br /><br /> Methods: Topographical thickness of the cornea was measured using OCT in all studies. The CRT^TM studies investigated myopic and hyperopic treatment, throughout the day. The myopic studies followed lens wear over a 4 week period, which was extended to 12 months, and investigated the thickness changes produced by two lenses of different oxygen transmissibility. CRT^TM for hyperopia (CRTH^TM) was evaluated after a single night of lens wear. <br /><br /> In the investigation of keratoconus, OCT corneal thickness values were compared to those obtained from Orbscan II (ORB) and ultrasound pachymetry (UP). A new fixation device was constructed to aid in the measurement of topographical corneal and epithelial thickness along 8 directions of gaze. Pachymetry maps were produced for the normal non-lens wearing cornea, and compared with the rigid gas permeable (RGP) lens wearing cornea and the keratoconic cornea. <br /><br /> Thickness changes prior to, and following LASIK were measured and monitored throughout six months. Myopic and hyperopic correction was investigated individually, as the laser ablation profiles differ for each type of procedure. The LASIK flap interface was also evaluated by using light backscatter data to monitor healing. <br /><br /> Results: Following immediate lens removal after myopic CRT^TM, the central cornea swelled less than the periphery, with corneal swelling recovering to baseline levels within 3 hours. The central epithelium decreased and mid-peripheral epithelium increased in thickness, with a more gradual recovery throughout the day. There also seemed to be an adaptation effect on the cornea and epithelium, showing a reduced amount of change by the end of the 4 week study period. The thickness changes did not alter dramatically during the 12 month extended study. In comparing the two lens materials used for myopic CRT^TM (Dk/t 91 vs. 47), there were differences in stromal swelling, but no differences in the central epithelial thinning caused by lens wear. There was a statistically insignificant asymmetry in mid-peripheral epithelial thickening between eyes, with the lens of lower Dk causing the greater amount of thickening. Hyperopic CRT^TM produced a greater increase in central stromal and central epithelial thickness than the mid-periphery. Once again, the stroma recovered faster than the epithelium, which remained significantly thicker centrally for at least six hours following lens removal. <br /><br /> Global pachymetry measurements of the normal cornea and epithelium found the periphery to be thicker than the centre. The superior cornea and epithelium was thicker than the inferior. In the measurement of the keratoconic cornea, OCT and ORB correlated well in corneal thickness values. UP measured greater values of corneal thickness. The keratoconic epithelium was thinner than normal, and more so over the apex of the cone than at the centre. The location of the cone was most commonly found in the inferior temporal region. Central epithelial thickness was thinner in keratoconics than in RGP lens wearers, which in turn was thinner than in non-lens wearers. <br /><br /> Following LASIK surgery for both myopia and hyperopia, the topographical OCT thickness profiles showed stromal thinning in the areas of ablation. The central myopic cornea showed slight regression at 6 months. During early recovery, epithelial thickness increased centrally in hyperopes and mid-peripherally in myopes. By the end of the 6 month study, mid-peripheral epithelial thickness was greater than the centre in both groups of subjects. The light backscatter profiles after LASIK showed a greater increase in backscatter on the anterior side of the flap interface (nearer the epithelium), than the posterior side (in the mid-stroma) during healing. The flap interface was difficult to locate in the OCT images at 6 months. <br /><br /> Conclusion: All the CRT^TM lenses used in this project produced more corneal swelling than that seen normally overnight without lens wear. In order for these lenses to be worn safely for long periods of time without affecting the health of the cornea, they need to be manufactured from the highest oxygen transmissible material available. The long-term effect of thinning on the epithelium's barrier properties needs to be monitored closely. <br /><br /> Global topographical thickness of the cornea and epithelium was measured using OCT in normal, RGP lens wearing and keratoconic eyes. Corneal and epithelial thickness was not symmetrical across meridians. The epithelium of RGP lens wearers was slightly thinner than normal, but not as thin as in keratoconics, suggesting that the epithelial change seen in keratoconus is mainly due to the condition. <br /><br /> Post-LASIK corneal and epithelial thickness profiles were not the same for myopic and hyperopic subjects, since the ablation patterns vary. Epithelial thickening in the mid-periphery had not recovered by six months in myopes or hyperopes, possibly indicating epithelial hyperplasia. Light backscatter profiles were used to monitor the recovery of the LASIK flap interface, showing the band of light backscatter around the flap interface to decrease as the cornea healed.

Background: Today corneal crosslinking with ultraviolet-A photoactivation of riboflavin is an established method to halt the progression of keratoconus. In some cases, when the refractive errors are large and the visual acuity is low, conventional corneal crosslinking may not be sufficient. In these cases it would be desirable with a treatment that both halts the progression and also reduces the refractive errors and improves the quality of vision. Aims:  The aims of this thesis were to determine whether mechanical compression of the cornea during corneal crosslinking for keratoconus using a sutured rigid contact lens could improve the optical and visual outcomes of the treatment, and also to find methods to evaluate the effect of different corneal crosslinking treatment regimens. Methods: In a prospective, open, randomized case-control study, 60 eyes of 43 patients with progressive keratoconus, aged 18-28 years, planned for routine corneal crosslinking, and a corresponding age- and sex-matched control group was included. The patients were randomized to conventional corneal crosslinking (CXL; n=30) or corneal crosslinking with mechanical compression of the cornea during the treatment (CRXL; n=30). Biomicroscopy, autorefractometry, best spectacle corrected visual acuity, axial length measurement, Pentacam® HR Scheimpflug photography, pachymetry, intraocular pressure measurements and corneal biomechanical assessments were performed before treatment (baseline) and at 1 month and 6 months after the treatment. One of the articles evaluated and compared the optical and visual outcomes between CXL and CRXL, while the other three articles focused on methods to evaluate treatment effects. In Paper I, the corneal light scattering was manually quantified from Scheimpflug images throughout the corneal thickness at 8 measurements points, 0.0 to 3.0 mm from the corneal centre, in patients treated with CXL. In Paper IV the corneal densitometry (light scattering) was measured with the Pentacam® HR software, in 4 circular zones around the corneal apex and at 3 different depths of the corneal stroma, in both CXL and CRXL treated corneas. Paper III quantified the biomechanical effects of CXL in vivo. Results: Corneal light scattering after CXL showed distinctive spatial and temporal profiles and Applanation Resonance Tonometry (ART) -technology demonstrated an increased corneal hysteresis 1 and 6 months after CXL. When comparing the refractive and structural results after CXL and CRXL, CRXL failed to flatten the cornea, and the treatment did not show any benefits to conventional CXL treatment, some variables even indicated an inferior effect. Accordingly, the increase in corneal densitometry was also less pronounced after CRXL. Conclusions: Analysis of corneal light scattering/densitometry shows tissue changes at the expected treatment location, and may be a relevant variable in evaluating the crosslinking effect. ART -technology is an in vivo method with the potential to assess the increased corneal hysteresis after CXL treatment. By refining the method, ARTmay become a useful tool in the future. Unfortunately, CRXL does not improve the optical and visual outcomes after corneal crosslinking. Possibly, stronger crosslinking would be necessary to stabilize the cornea in a flattened position.

Syfte: Att hitta de senaste behandlingsmodaliteterna som finns tillgängliga för behandling av keratoconus genom att genomföra en litteraturstudie. Metod: En enkel sökfråga för keratoconusbehandling gjordes i databasen av National Library of Medicine Website (pubmed.gov). Olika filter användes för att få resultat inom de senaste 5 åren. De var uppdelade enligt ämnet för varje artikel i tre kategorier: Icke-kirurgisk hantering, kirurgisk hantering och strategier för hantering och protokoll av keratoconus. Resultat: Totalt 459 artiklar erhölls. Av alla dessa valdes 18 ut för den nuvarande studie. Fyra artiklar var i den icke kirurgisk kategori, 12 artiklar i kirurgisk kategori, medan två var i kategorin om strategier för hantering och protokoll av keratoconus. Icke-kirurgiska modaliteter för behandling av mild till måttlig keratoconus har fortfarande stor inverkan, särskillt med tillkomsten av nya hybridlinser. Kirurgisk behandling av avancerade fall av keratoconus ger fortfarande bra resultat. Utvecklingen av hanteringsprotokollet och hanteringsstrategier har nått nya fronter baserade på de olika behandlingsalternativen som finns tillgänliga idag. Slutsats: En genomgripande förbättring kan ses vid behandling av keratoconus de senaste åren, tack vare nya tekniker och utveckling av gamla. Optiker tillsammans med ögonläkare har sin roll i att förbättra livskvaliteten hos keratoconus patienter.

Two corneal dystrophies, posterior polymorphous corneal dystrophy (PPCD) and keratoconus, have been associated with missense mutations found in the transcription factor-encoding gene Visual System Homeobox 1 (VSX1). Despite this association, the pathogenic link between VSX1 and these diseases remains controversial. To address this issue, I utilized a variety of in vitro approaches to study how seven VSX1 missense mutations found in disease populations that span two highly conserved domains, the homeodomain (HD) and CVC domain affect VSX1 transcriptional activity, protein expression levels and subcellular localization. I also carried out an in vivo investigation by generating a mouse line carrying a mutation in Vsx1: P254R. Corneal morphology was examined through histology and ex vivo whole eye confocal imaging which was used to assess corneal thickness. Quantification of immunocytochemistry was used to characterize terminal marker expression in the inner retina compared to previously described phenotypes in Vsx1-null mice. My in vitro results showed that mutations found in both the HD and CVC domain alter the normal transcriptional repression activity in Vsx1. These changes were not due to changes to protein expression or subcellular localization. Characterization of corneal and retinal phenotypes in vivo revealed no significant differences in Vsx1 P254R mice when compared to wild-type and Vsx1-null controls. In conclusion, my work shows that Vsx1 P254R is not pathogenic for corneal dystrophies in a mouse model. However, my in vitro studies show that Vsx1 mutations have the ability to alter transcriptional activity and therefore still have the potential to be pathogenic in humans. Further investigation is needed to determine whether VSX1 mutations found in disease populations are, in fact, causative for corneal dystrophies. / Graduate / 2019-04-25

Brittle cornea syndrome (BCS) is an autosomal recessive, multisystemic connective tissue disorder characterised by extreme corneal thinning and fragility. Mutations in transcription factors ZNF469 and PRDM5 cause BCS types 1 and 2, respectively. Both genes are believed to regulate the transcription of extracellular matrix (ECM) components, particularly fibrillar collagens, and are suggested to act on a common pathway. Molecular diagnosis is available for affected patients, and those at risk of being heterozygous carriers. Chapter 3 presents the identification of mutations in ZNF469 in 14 families with BCS, and evidence for the downregulation of ECM-associated transcripts in skin fibroblasts from patients with ZNF469-associated disease by Q-PCR.Chapters 4 and 5 focus on PRDM5-associated disease. Chapter 4 highlights previously undescribed and potentially phenotype-related aspects of PRDM5- associated BCS. In chapter 4, a potential role for PRDM5 in development of Bruch’s membrane is suggested, by the observation of significantly reduced expression of major components of Bruch’s membrane, including collagens types I, III, and IV in patients with PRDM5-associated disease using immunohistochemistry. A first description of PRDM5 expression in the human eye is also presented in chapter 4. In chapter 5, a potential role for PRDM5 in retinal vasculogenesis is suggested. PRDM5-related disease also offers an in vivo opportunity to observe a subset of epigenetic regulatory mechanisms in an inherited eye disease, providing mechanistic insights, presented in chapter 5. Examination of PRDM5 interaction partners by pull-down and mass spectrometry reveals the diminished interaction of a PRDM5 construct carrying a BCS-associated mutation with repressive complexes, and, through studies on fibroblasts and retinal tissue from patients, we suggest a role for dysregulation of the repressive histone mark H3K9 di- methylation in vivo. These findings suggest a role for a molecular network surrounding dysregulated H3K9 di-methylation in PRDM5-associated disease. Finally, chapter 6 expands the study of a rare disease into more common diseases investigating the role of genetic variations in ZNF469 and PRDM5 in keratoconus, an ocular disorder resulting in progressive corneal thinning. I identified enrichment of rare potentially pathogenic alleles in ZNF469 in 12.5% of keratoconus patients, highlighting ZNF469 as the most significant genetic factor responsible for keratoconus identified to date. In conclusion, this study of a rare disease, BCS, has provided translational research insights (chapter 3), functional insights (chapter 4) mechanistic insights (chapter 5) and has expanded into other, less rare, diseases (chapter 6).

Bakgrund Kornea är det högsta brytande mediet i ögat där små skillnader i dess form kan ändra brytkraften vilket leder till kornealastigmatism. Detta är viktigt att kunna mäta för synkorrektion och behandling av sjukdomstillstånd.    Syfte Syftet med studien var att undersöka repeterbarhet och jämförbarhet för de tre instrumenten Essilor WAM700+, Topcon TRK-2P och CSO/BON Sirius gällande kornealastigmatism.   Metod Denna kvantitativa tvärsnittsstudie hade bara ett urvalskriterie vilket var att deltagarna skulle vara utan kända patologiska ögontillstånd. Minst tre mätningar per deltagare utfördes per instrument utefter ett framtaget protokoll.   Resultat Majoriteten av de 64 personer som undersöktes var &lt;50 år och 122 ögon ingick i analyserna då några uteslöts p.g.a. inkompletta resultat. Det fanns inga signifikanta skillnader mellan instrument gällande J180 och Power scalar-komponenter, däremot fanns en signifikant skillnad för J45-komponenten. ICC-värdena för samtliga datagrupper blev &gt;0,90, vilket visar på hög repeterbarhet. CR hamnade för P-komponenten på ~0,50DC för samtliga instrument.   Slutsats Resultaten av denna studie visar att vid fallet av friska ögon krävs mer än en mätning för att undersöka kornealastigmatism. Skillnaden mellan instrumenten i studien är bara kliniskt signifikant vid sned astigmatism. / Introduction Cornea is the medium with the highest power of refraction in the eye. This means that small differences in the curvature affects the refractive power which can lead to corneal astigmatism. Measuring of corneal astigmatism is essential in some cases for finding the correct prescription and treatment of corneal dystrophies. Purpose The purpose of this study was to examine the repeatability and comparability of Essilor WAM700+, Topcon TRK-2P and CSO/BON Sirius when it comes to corneal astigmatism. Methods This quantitative, cross-sectional study’s only exclusion criteria was that the participants did not have any known pathological eye conditions. At least three measurements were made per participant per machine according to a protocol.   Results The majority of the 64 patients in the study were &lt;50 years of age. In total 122 eyes were used in analysis after the exclusion of cases with incomplete data. No significant difference was found for either the J180 or the power scalar component, whereas the J45-compontent showed a significant difference. The ICC-values for all data groups were calculated to &gt;0,90 which means the repeatability is good. The CR for P in all instruments was calculated to ~0,50DC.    Conclusion The results of this study shows that, in healthy eyes, more than one measurement is necessary in measuring corneal astigmatism. The differences found between the instruments are only clinically significant for oblique astigmatism.

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The extracellular matrix (ECM) offers a protective shelter for cells and provides signaling paths important for cell to cell communication. ECM consists of basement membranes (BM) and interstitial matrix. BMs provide mechanical support for parenchymal cells, influence cell proliferation, survival, migration and differentiation. They are also important for tissue integrity. Laminins (LM) are the major non-collagenous component of BMs. Cell-ECM interactions, mediated by receptors, are indispensable during embryonic development, wound healing, remodeling and homeostasis of tissues. The integrins are the major cell-adhesion receptors. The expression of alpha11 integrin chain in the cornea is of great interest, as it is part of the alpha11beta1 integrin receptor for collagen type I, the predominant component of the corneal stroma. The aims were to thoroughly characterize the ECM in the developing and adult human eye, with particular focus on the cornea, LM and alpha11 integrin chains, and to examine alpha11 integrin chain in an animal model of corneal wound healing and remodeling. Human fetal eyes, 9-20 weeks of gestation (wg), and adult human corneas with different diagnosis were treated for immunohistochemistry with specific antibodies against LM and alpha11 integrin chains. Normal and knockout (ko) mice were treated with laser surgery to create a deep wound in the corneal stroma. The wound healing process was followed at different time points. The cellular source of alpha11 integrin chain was studied in cell cultures. In the fetal eyes, the BM of the corneal epithelium, the Descemet’s membrane (DM) and the Bruch’s membrane each had their specific combinations of LM chains and time line of development, whereas the lens capsule and the internal limiting membrane showed constant LM chain patterns. The epithelial BMs of normal and diseased adult corneas contained similar LM chains. The normal morphology of the epithelial BM was altered in the different diseases, particularly when scarring was present. In the scarred keratoconus corneas there were excessive LM chains. The majority of keratoconus corneas also expressed extra LM chains in the DM. At 10-17 wg alpha11 integrin chain was present in the human corneal stroma, especially in the anterior portion, but it was scarce at 20 wg, in normal adult corneas and in Fuchs’ endothelial dystrophy. In contrast, it was increased in the anterior portion of the stroma in keratoconus corneas with scarring. Alpha11 integrin ko mice had a defective healing with subsequent thinner corneas. Alpha11 integrin expression correlated to the presence of alpha-smooth muscle actin in vivo as well as in vitro. The distinct spatial and temporal patterns of distribution for alpha11 integrin and each of the LM chains suggest that they play an important role in human ocular differentiation. The selectively affected LM composition and the novel expression of alpha11 integrin chain in scarred keratoconus corneas as well as the pathologic healing in ko mice, indicate that alpha11 integrin and LM chains also play an important role in the process of corneal healing, remodeling and scarring and might participate in the pathogenesis of corneal disease. This knowledge is of practical importance for future topical therapeutic agents capable of modulating the corneal wound healing processes.