| 1. |
- Al-Hawasi, Abbas, 1976-
(författare)
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Retinal ganglion cell examination with Optical Coherence Tomography reflects physiological and pathological changes in the eye and the brain.
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The retinal ganglion cell is situated in the inner retina and its axons, composing the retinal nerve fiber layer (RNFL), leave the eye to form the optic nerve. These cells develop embryologically from the forebrain and later during development re-establish connections with different parts of the brain serving different purposes. This unique position and connections make it possible to be investigated with different methods. Optical Coherence Tomography (OCT) is an accessible and easily operated clinical device that can provide a detailed image of this layer at a few micrometers level of precision in measurements. In this thesis we aimed to see whether examining these cells with OCT could reflect physiological and pathological changes in the eye and brain.In cases of optic neuritis (Paper I), the OCT examination showed early thickening of the peripapillary (pRNFL) followed by thinning which takes 6-9 months to reduce to below normal thickness without the ability to distinguish between the real from pseudo thinning. The ganglion cell -inner plexiform layer (GCL-IPL) layer, however, showed a thickness reduction within a few weeks to 3 months without pseudo thinning. In cases of Idiopathic Intracranial Hypertension (IIH) (Paper II), the GCL-IPL remained unchanged and there was no difference in pRNFL thickness compared to healthy controls, whereas the optic disc parameters of rim thickness, rim area, cup volume and cup/disc ratio differed significantly (P<0.05).In cases of benign multiple sclerosis (Paper IV), the OCT could detect that eyes which are not affected by optic neuritis had an annual thinning rate of the RNFL and GCL-IPL similar to a healthy population (P>0.05) which may indicate the benign course of the disease. In cases of physiological factors affecting the GCL in healthy population (Paper III) the OCT examination showed that there was a significant thinning rate of the layer with age (P<0.05), but the thinning was not significant when sex and axial length of the eye were taken into consideration. Males had a thicker GCL volume than females and with age a significant reduction in GCL volume was noted in females but not in males. A Longer axial length of the eye found to be associated with thinner GCL volume. In conclusion retinal ganglion cell changes detected with OCT can reflect physiological and pathological changes in the eye and brain.
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| 2. |
- Xeroudaki, Maria, 1989-
(författare)
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Advanced surgeries, medicines and materials for corneal regeneration
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Corneal transplantation is often the only treatment option in cases of corneal blindness, with the main challenges being the scarcity of human donors, risk for graft failure and suboptimal visual outcome due to suture-related issues. Alternative therapies are sought that either address the above transplantation issues directly or stimulate the cornea’s repair mechanism and regenerative properties to restore transparency without the need for transplantation. The first aim of this thesis was to develop a cell-free substitute for the human corneal stroma made of porcine collagen, a purified byproduct from the food industry that is already approved by FDA as a raw material, used for example in cosmetic surgery or as a medical device in glaucoma surgery. Abundance, cost-effectiveness, low rejection risk due to acellularity, high purity and worldwide availability are among the main advantages of purified porcine collagen compared to human donors and other corneal stromal substitutes. The second aim was to address the risk of graft rejection in cases of neovascularized and inflamed corneas by loading the bioengineered porcine collagen constructs with drugs that can ideally promote corneal regeneration, secure the survival of the graft in cases of high-risk keratoplasties and minimize the need for prolonged topical immunosuppressive therapy following operation, which has drawbacks of low bioavailability and need for good patient compliance. The third aim was to develop alternative techniques of lamellar transplantation, all assisted by femtosecond laser, that are less suture-dependent and enable the intrastromal implantation of biomaterials. Finally, the fourth aim of this thesis was to evaluate the role of Regenerating Agent (RGTA) eye drops in corneal wound healing following therapeutic laser ablation of the cornea in a randomised, blinded, placebo-controlled preclinical study. Regenerating Agent is a biomimetic of extracellular matrix with well-established favourable outcomes in the treatment of skin wounds and preliminary positive results in the treatment of corneal wounds. Different manufacturing protocols were used to enhance the mechanical properties of bioengineered porcine collagen (BPC) and to address different requirements. The combination of both chemical and photochemical crosslinking with riboflavin and ultraviolet A light (to form BPCDX) and reinforcement with cellulose nanofibers extracted from the Ciona intestinalis sea invertebrate (BPCDX-CNF) resulted in stronger biomaterials compared to earlier BPC versions. The biomaterials could be manufactured in different sizes and in core-and-skirt forms with the peripheral skirt degrading faster due to mechanical compression without the addition of any cross-linkers during manufacturing. BPC could be successfully loaded with nerve growth factor (NGF) and BPCDX-CNF with dexamethasone without sacrificing transparency and both drugs could be released from BPC-based materials in vitro up to at least 2 months. The biological activity of dexamethasone released from the drug-loaded BPCDX-CNF could be confirmed by the decreased expression of inflammatory cytokines in human corneal epithelial cells grown on dexamethasone-loaded BPCDX-CNF. A compatible packaging and sterilization process was developed for BPCDX, tested internally and externally by Good Laboratory Practice-certified laboratories, that can enable worldwide distribution and storage at room temperature or in a refrigerator up to two years without the need of extra quality controls before transplantation. BPC-based materials could be safely implanted in rabbit, minipig and human corneas with advanced keratoconus using femtosecond-laser assisted intrastromal keratoplasty procedures. A femtosecond laser was used to create intrastromal pockets of different dimensions based on the size of the biomaterials. Through an access cut or by lifting a flap, both created by femtosecond laser, biomaterials could be implanted intrastromally with or without native tissue removal. Additional sutures were used in a surgical inflammatory model to test the biological activity of dexamethasone released by BPCDX-CNF. These minimally invasive surgical procedures required shorter period of immunosuppression following operation and maintained the anatomy of the surrounding host tissue. Apart from the skirt part of the composite BPC that was mechanically compressed and was designed to degrade faster, the crosslinked BPC remained stable in animal models, while no degradation was observed in the BPCDX 2 years after implantation in humans. The biomaterials were biocompatible and native cells were found in the biomaterial-host interface or in the periphery of the biomaterial. The inflammatory response following operation depended on individual response to injury and did not appear to be stimulated by BPC implantation. Neovascularization and haze formation were mainly restricted around the sutures used to secure either the access cut or flap overlying the biomaterials as well as intentionally placed close to the limbus to trigger inflammation in the dexamethasone study. In the human studies, no sutures were used and corneal transparency was maintained at the highest level in all subjects after 2 years without any signs of rejection, inflammation, vascularization or scarring. Topographic indices including mean anterior corneal curvature and maximal corneal apical curvature were significantly reduced in both clinical cohorts resulting in improved best corrected visual acuity. Importantly, following operation all human subjects could tolerate contact lenses and no subject was considered legally blind. The release of dexamethasone from the drug-loaded BPCDX-CNF could be also confirmed in vivo by sustained intraocular pressure increase, tendency to reduce neovascularization and haze formation and sustained suppression of inflammatory cytokines in the aqueous humor of eyes implanted with dexamethasone-loaded BPCDX-CNF compared to non-loaded BPCDX-CNF. Finally, RGTA eye drops following excimer laser ablation of the anterior healthy rabbit cornea did not affect the already quick and uneventful epithelial closure. Although there was significantly less haze in the RGTA group compared to placebo as measured by in vivo confocal microscopy, this decrease was not clinically relevant as all corneas in both groups were clinically transparent following laser. The microscopic haze formation and staining problems did not allow quantification of nerve regeneration, but subbasal nerves were found to repopulate the central ablated regions in both RGTA and placebo groups. In conclusion, our results indicate that biomaterials made of bioengineered porcine collagen are a safe alternative to human donor tissue for corneal transplantation, offering the advantage of custom-made manufacturing to address different requirements with potential applications to different corneal stromal diseases. Femtosecond laser enables safe intrastromal implantation of biomaterials with less suture-related issues and immunosuppression following operation compared to traditional corneal transplantation methods. Regenerating Agent eye drops do not affect the already rapid wound healing following laser ablation of the healthy cornea.
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| 3. |
- Germundsson, Johan
(författare)
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Surgical outcomes of phototherapeutic keratectomy on Epithelial basement membrane dystrophy, and the characterisation of Bowman´s Layer
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background. Epithelial basement membrane dystrophy (EBMD) is a common disease of the anterior cornea that can lead to problems with vision and/or painful recurrent erosions of the corneal epithelium. Several treatment options have been used, but recurrence of EBMD after treatment is a problem. Excimer laser phototherapeutic keratectomy (PTK) has become an increasingly popular surgical option in recent years due to its accuracy, reproducibility, and good clinical outcomes. When treating EBMD with PTK, the anterior corneal structures including the epithelium, Bowman´s layer (BL), and subbasal nerves are disrupted or removed completely. Little is known about how BL, nerves, and the stroma recover after PTK treatment, or how they could influence recurrence of EBMD symptoms. Additionally, very little is known about the properties and actual thickness of BL in-vivo.Aims. To improve the understanding and management of EBMD by investigating the clinical diagnosis and treatment of EBMD and its relationship to Bowman´s layer.Method. An excimer laser was used to treat EBMD patients at the Department of Ophthalmology during the period 2001-2010. IVCM was used to perform pre- and postoperative examinations. In particular, images of anterior corneal structures, cells, and nerves in high-resolution were obtained. Additionally, a group of over 100 healthy volunteers underwent a full ophthalmic examination including IVCM. Other subjects examined in this work included a group of 17 patients who underwent full-thickness transplantation of the cornea.Results and conclusions. Clinical follow-up revealed that PTK is an effective method of alleviating the clinical symptoms of EBMD, but the dystrophy can recur with time. Recurrence can be divided into clinical and morphologic types, and may depend upon treatment parameters including the type and depth of ablation. IVCM was found to be a useful screening tool pre- and postoperatively, and could prevent patients with symptoms, but no visible signs of EBMD on slit lamp examination, to go undiagnosed and untreated. BL was found to play a role in regenerative wound healing after PTK, and was also found to be important regarding the treatment and recurrence of EBMD. BL may present a physical barrier that protects the subepithelial nerve plexus thereby facilitating sensory recovery, and BL may also serve as a barrier that prevents direct traumatic contact with the corneal stroma, avoiding a stromal wound healing response. To aid in accurate assessment of BL in patients, an in vivo method for determining BL thickness was developed. This method could be an important tool to aid in clinical assessment and planned treatments of the anterior cornea. Using this tool, a large inter-individual variability in BL thickness and a strong negative correlation of BL thickness with age were found in a healthy population. Using IVCM, it was also found that subbasal nerves are pathologically reduced in EBMD compared to a healthy population, and that this nerve deficit does not improve in the long term after PTK treatment.
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| 4. |
- Kulikovska, Marina
(författare)
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Corneal stromal cell responses to traumatic wounds and topical treatments
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background. The cornea has unique anatomic, cellular, molecular, and functional features that lead to important mechanistic differences in the process of repair in comparison with what occurs in skin and other organs. The first observable stromal response in corneal wound healing is keratocyte apoptosis. Shortly thereafter, remaining keratocytes in adjacent areas obtain a fibroblastic phenotype and begin to proliferate and to migrate, transforming into myofibroblasts, a phenotype associated with remodeling of stromal collagen. Return to normalcy following wound healing includes elimination of myofibroblasts and restoration of the quiescent state of the keratocytes. Often, however, a wound healing response results in the persistence of myofibroblasts and their subsequent production of fibrous scar tissue.Aims. The overall aim is to understand the role of keratocytes, and their phenotypic variations in a cornea subjected to various types of trauma or treatments. More specific aims are to define expression pattern of alpha-smooth muscle actin (α-SMA) and chaperonin containing T-complex polypeptide 1 (CCT) in ultraviolet radiation wound model, to evaluate the effect of biglycan and platelet rich plasma (PRP) treatment during wound healing after corneal incision, and to characterize the structure of the bioengineered porcine construct and its interaction with stromal cells after implantation.Methods. CCT and α-SMA expression level was evaluated by reverse transcription polymerase chain reaction (RT-PCR) in rabbit corneas subjected to ultraviolet radiation (UVR). Effect of biglycan and PRP on keratocyte phenotype and survival was evaluated by immunohistochemistry, and real time PCR using rat corneas after incisional wounding. Bioengineered porcine construct (BPC) was implanted into rabbit corneas using femtosecond laser-enabled intrastromal keratoplasty (FLISK) and characterized by means of immunohistochemistry, electron microscopy, and in vivo confocal microscopy (IVCM).Results and conclusions. In a mild wound, the expression of α-SMA mRNA is followed by expression of mRNA of at least one subunit of the complex folding α-SMA. At protein level, α-SMA is detected in the front line of repopulating keratocytes. Expression levels for both mRNAs decline as the stroma repopulation process progresses.Biglycan appears to accelerate corneal wound healing in vivo by modulating myofibroblast apoptosis, resulting in removal of myofibroblasts that may otherwise compromise corneal transparency.PRP treatment resulted in suppressed stromal cell apoptosis followed by SMAD3 activation and a greater proportion of myofibroblasts present at the wound site. Suppression of stromal cell apoptosis after corneal wounding by use of a growth factor rich formulation may lead to myofibroblast accumulation by modulation of the TGF-β pathway.A cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate normal regenerative healing of the corneal stroma.Taken together, results present an interesting possibility to combine BPC implantation and topical biglycan treatment to improve surgical outcome in future studies.
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| 5. |
- Bourghardt Peebo, Beatrice, 1968-
(författare)
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Angiogenesis from a new perspective
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Angiogenesis is the emergence of new blood and lymph vessels from existing ones. In the pathologic form it contributes to the onset and progression of numerous different human disorders such as cancer, inflammation, atherosclerosis and blinding eye diseases. There exist a number of models to study angiogenesis, both in vitro and in vivo, but there is no single perfect model so far. Consequently there is a need to develop new angiogenesis assays for evaluating blood and lymph vessel behaviour in different physiologic settings.The aim of this thesis was to gain insight into in vivo angiogenesis introducing a new technique in an inflammatory corneal model. The method involved in vivo examination of the cornea and subsequent comparison of in vivo findings with ex vivo immunohistochemical analysis of the same tissue samples. An existing suture model for inflammatory angiogenesis in the cornea was modified for in vivo observations with a clinically-approved corneal confocal microscope.In this thesis, corneal lymph vessels were characterized for the first time in vivo and findings from the experimental bench could be applied in a clinical setting, where presumed lymphatics were observed in a corneal transplant patient with rejection. Furthermore, the technique was extended to investigate time-lapse processes in sprouting and regressing capillaries, and led to a number of new observations. CD11b+ myeloid cells constitute the first bulk of infiltrating inflammatory cells and contribute to inflammatory sprouting and regression in numerous ways including pre-patterning of the corneal stroma and guiding of capillary sprouts. Newly formed hemangiogenic sprouts are perfused with a slow-moving fluid and have a lumen. In blood vessel regression, capillary remodeling occurred by abandonment of sprout tips in close association with macrophages and vascular loops formed by presumed intussusceptive angiogenesis. In addition, a network of pericyte- and endothelium-free basement membrane tubes was formed after desertion or degradation of vascular endothelium in former corneal capillaries.In conclusion, we introduce a new in vivo technique for investigating angiogenesis in a corneal model were in vivo findings can be interpreted with ex vivo definitions of specific cell types by immunohistochemistry. Findings from pre-clinical experiments have been possible to apply in a clinical setting when examining patients with corneal pathology.
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| 6. |
- Hammar, Björn, 1963-
(författare)
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Two New Corneal Diseases Characterized by Recurrent Erosions
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Recurrent corneal erosions are a common complication of superficial corneal wounds. They most commonly arise following a trauma, in association with various corneal dystrophies, or are idiopathic.The main aim of this thesis was to investigate two hereditary corneal diseases with recurrent erosions in order to find out if they had been described before, and more specifically to describe the clinical picture and the morphological changes, differentiate them from other known autosomal dominant corneal dystrophies with a clinical resemblance, and to exclude genetic linkage to known corneal dystrophies with autosomal-dominant inheritance and a clinical resemblance.The thesis is based on two families of subjects belonging to different phenotypes. The subjects from Småland (Dystrophia Smolandiensis) belonged to a six-generation family, which included 171 individuals of whom 44 were affected individuals, and the family from Hälsingland (Dystrophia Helsinglandica) included sevengenerations of 342 individuals, of whom 84 were affected. The individuals in both families were investigated by collection of medical history through medical records and questionnaires assessing different aspects of the diseases, pedigree analysis, and from clinical examination. Haplotype analysis was used to exclude genetic linkage of both diseases to known autosomal-dominant corneal dystrophies with a clinical resemblance. The morphological changes in Dystrophia Smolandiensis were investigated by examining affected individuals with in-vivo confocal microscopy and/or slit-lamp biomicroscopy, and examining corneal tissue samples using histopathology and immunohistochemistry. In Dystrophia Helsinglandica, the morphological changes were described using in-vivo confocal microscopy and/or slit-lamp biomicroscopy, but also using videokeratography and corneal sensitivity measurement.The main results were the findings of two new corneal disorders with autosomal dominant inheritance, characterized by recurrent corneal erosions.In Dystrophia Smolandiensis the symptoms often started within the first year of life. The number of recurrences per year was highest from the onset and for about 30-40 years, and the duration of recurrence could stretch up to 21 days. The frequency of recurrences was variable in the disease from continuous symptoms to once a year and tended to decrease later in life. The risk of having recurrences did not disappear completely with age. Typical precipitating factors of recurrence were draught and a common cold. About two thirds of the affected individuals responded well to oral vitamin B treatment, but no other therapy has so far been successful. In Dystrophia Smolandiensis development of corneal opacifications or secondary scarring of varying type and degree was seen in about half of the subjects. Opacifications were first noted at the age of about 7 years, but usually first seen at the age of 20-40 years. Corneal grafting was performed in nine individuals, and recurrences were seen in all grafts. The corneal buttons showed epithelial hyperplasia, partial or total loss of Bowman’s layer, and subepithelial fibrosis in the light microscope. The deeper stroma, Descement’s membrane, and endothelium were normal. Confocal microscopy confirmed loss of Bowman’s layer and revealed that the corneal nerves either were normal in their sub-basal plexa or showed signs of regeneration. None of the morphological findings were specific. We believe that the opacifications are reactive corneal changes to repeated erosive events.The onset in Dystrophia Helsinglandica was usually at the age of 4-7 years and late-developing subepithelial fibrosis not significantly affecting visual acuity was seen in all affected individuals over the age of 37 years. The number of recurrences per year was highest from the onset and for about 20-30 years, and the duration of recurrence was usually up to about a week. The frequency of recurrences tended to decrease in the disease with increasing age, but did not cease completely. The precipitating factor of recurrence was typically a minor trauma. No therapy has so far been successful in the family. The corneal changes of affected individuals were classified into different stages from a nearly normal cornea to progressive fairly discrete subepithelial fibrosis of the central cornea. Discrete localized Subepithelial fibrosis in the periphery or mid-periphery (stage I) was the sole finding in 12% of the individuals. A more widespread subepithelial fibrosis, mainly in the mid-periphery, was found in 31% of the individuals (stage II). In stage III, the subepithelial fibrosis engaged the central cornea but did not affect the vision to a significant degree. In late phases of stage III small jellylike corneal irregularities could be seen. We believe that the opacifications are reactive changes to repeated erosive events.In conclusion this thesis describes two new corneal disorders – Dystrophia Smolandiensis and Dystrophia Helsinglandica.
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