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Processing and analysis of ophthalmologic images and data
Brož, Petr ; Hozman, Jiří (referee) ; Kolář, Radim (advisor)
In this work is describe anatomy and physiology of the cornea. The following are the primary non-inflamatory degeneration of the cornea. Then describe the physical principles diagnostic devices for cornea – keratometer, pachymeter, Michelson interferometr and optical coherence tomography (OCT). At the end of the theoretical introduction is describes the principle of laser correction surgery – LASIK. The practical part is divided into two main objectives. The first task is propose an algorithm for automatic detection of corneal surface and then calculation of corneal thickness and size of the chamber angle in Matlab. The aim of the second task is image flap analysis for boundary detection.
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Immunomodulatory and differentiation properties of MSCs in a mouse model of the injured cornea and retina
Kössl, Jan ; Holáň, Vladimír (advisor) ; Vištejnová, Lucie (referee) ; Heissigerová, Jarmila (referee)
Stem cells, in general, represent the potential for treating many diseases and disorders that are currently difficult to treat or the therapy has many side effects. One of the stem cells widely investigated these days are mesenchymal stem cells (MSCs). MSCs have the considerable immunomodulatory and regenerative potential for treating degenerative disorders and severe damage to various parts of the eye or other organs. Likewise, their application could serve as supportive therapy in corneal transplantation and other eye inflammatory conditions. In this study of immunomodulatory properties of MSCs, we have focused mainly on their ability to differentiate into cells of different tissue types (in our case, corneal epithelium and retina), their production of immunomodulatory molecules in the inflammatory environment, their ability to migrate to the site of the injury, and their local anti-inflammatory, regenerative, and anti-apoptotic effects. In addition, we tested the therapeutic effects of MSCs in a mouse model of ocular surface injury and a model of retinal degeneration. Finally, we investigated the mechanism of this effect in in vitro models with explants of these tissues. Limbal stem cells (LSCs) are already used to treat severe corneal damage as limbal stem cell deficiency. However, this...
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The role and function of stromal enzymes in keratoconus pathogenesis
Ďuďáková, Ľubica ; Jirsová, Kateřina (advisor) ; Svozílková, Petra (referee) ; Ardan, Taras (referee)
Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...
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Expression of endogenic lectins and their glycoligands in the tear fluid, human corneal and conjunctival epithelium under physiological and disease conditions
Hrdličková, Enkela ; Filipec, Martin (advisor) ; Heissigerová, Jarmila (referee) ; Čejková, Jitka (referee)
Purpose: Lectins play an important role in many biological processes. The aim of this work was to analyse mainly the expression of endogenic lectins, such as galectins and plant lectin, e.g. Dolichos biflorus agglutinin (DBA), and their glycoligands in the tear fluid, human corneal and conjunctival epithelium in physiological and disease conditions. Further, we studied the human natural antibody against Galα1,3Gal-R, which is mainly responsible for hyperacute rejection of xenografts transplants. We tried to investigate its localization in human corneal epithelium, lacrimal gland and tears. Material and Methods: Human tissue (lacrimal gland, tear fluid, conjunctiva, cornea, epidermis, keratinocyte and cultured corneal epithelium), as well as porcine tissue (cornea, liver and epidermis) were examined. Endogenous galectins (galectins-1, -3 and -7) were detected using immunohistochemistry methods. Binding sites for galectins, as well as binding sites for plant lectin Dolichos biflorus agglutinin, were localized by lectin histochemistry. Reverse lectin histochemistry was used for the study of binding reactivity of endogenous lectins using labelled (neo)glycoligands. Employing biotinylated natural human IgG anti -galactosides, as well as anti -galactosides, we detected reactive epitopes in human...
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Phenotypical characterization of the healthy human cornea and the alterations caused by posterior polymorphous corneal dystrophy
Reinštein Merjavá, Stanislava ; Jirsová, Kateřina (advisor) ; Martínek, Jindřich (referee) ; Čejková, Jitka (referee)
Purpose: The aim of this work was to characterize the healthy human cornea and the cornea of patients suffering from posterior polymorphous corneal dystrophy (PPCD) using different antibodies. Despite the fact that PPCD is a very rare disorder, one of the largest groups of PPCD patients in the world comes from the Czech Republic. This offers us the opportunity to investigate the changes on the clinical, cellular and molecular levels. Material and Methods: A collection of 25 control corneas as well as 16 pathological corneas from PPCD patients were used. Epithelial (cytokeratins) and mesothelial markers (mesothelin, calbindin 2, HBME-1 protein) were detected in all layers of the healthy corneas using immunocyto- and immunohistochemistry. The expression of all markers was confirmed using molecular methods as well (RT-PCR and Western blot). Changes in the expression of cytokeratins and changes in the extracellular matrix structure (collagen IV and VIII) were studied in the PPCD corneas. Combined fluorescent immunohistochemistry with fluorescence in situ hybridization were used in order to characterize the origin of abnormal cells on the posterior graft surface, which cause the recurrence of the PPCD after penetrating keratoplasty surgery. Results: Changes in the cytokeratin expression (strong...
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Use of corneal endothelium and amniotic membrane for transplantation purposes.
Šmeringaiová, Ingrida ; Jirsová, Kateřina (advisor) ; Netuková, Magdaléna (referee) ; Čejková, Jitka (referee)
Part I: Endothelial cells form the posterior layer of the cornea and are important for maintaining its transparency. Dysfunctional endothelium can only be restored by transplantation. The global shortage of donor corneas requires the search for alternative treatments. The preparation of the graft by tissue engineering methods is complicated by low proliferative capacity of endothelium. To date, no endothelium-specific marker has been defined and the existence of endothelial stem cells has not been confirmed yet. We have prepared a protocol for culturing endothelial cells from research-grade tissue - corneoscleral rims obtained after transplantation or corneas excluded from the transplant process. We monitored localization of selected proteins, including stem cell markers, in native tissue and in primary cell cultures. We prepared up to 6.4 cm2 of endothelium from one cornea/rim, which had cellular features comparable to the native endothelium. This approach can increase the amount of endothelium for research or transplantation purposes. Using indirect immunohistochemistry, we showed that none of the previously proposed endothelial molecular markers is specific for these cells. We detected the expression of stem cell markers throughout the endothelial layer. In the porcine cornea model, we monitored...
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The culture of limbal and mesenchymal cells on various feeders for their use in ophthalmology.
Trošan, Peter ; Jirsová, Kateřina (advisor) ; Heissigerová, Jarmila (referee) ; Netuková, Magdaléna (referee)
P.Trošan Ph.D. Thesis Abstract Limbal stem cell deficiency (LSCD) is a disease characterized by the deficiency of stem cells in the limbus, which are responsible for the homeostasis and renewal of the corneal epithelium. This disorder results in corneal neovascularization, chronical inflammation and opacification, which may lead to loss of vision. The most successful treatment is the transplantation of limbal tissue or cultured limbal epithelial cells (LECs) onto the damaged ocular surface. The human amniotic membrane (HAM) is used as the feeder of the LECs culture, as well as for the LSCD treatment. HAM is also widely used in clinical practice, particularly for the treatment of chronic wounds. This dissertation is particularly concerned on cell therapy for LSCD, on preparation of cells suitable for grafting onto the ocular surface, on the improvement of the LECs culture conditions, and on the preparation of appropriate carrier for the transfer of cells onto the damaged cornea. During my work I have used a wide spectrum of methods, e.g. cell cultures (LECs, mesenchymal stem, amniotic epithelial, conjunctival epithelial, goblet and 3T3 cells), immunohisto- and immunocytochemistry, microscopy, proliferation and colony forming assays, reverse transcription and quantitative real-time PCRs and statistical...
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The role and function of stromal enzymes in keratoconus pathogenesis
Ďuďáková, Ľubica ; Jirsová, Kateřina (advisor) ; Svozílková, Petra (referee) ; Ardan, Taras (referee)
Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...
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Expression of endogenic lectins and their glycoligands in the tear fluid, human corneal and conjunctival epithelium under physiological and disease conditions
Hrdličková, Enkela ; Filipec, Martin (advisor) ; Heissigerová, Jarmila (referee) ; Čejková, Jitka (referee)
Purpose: Lectins play an important role in many biological processes. The aim of this work was to analyse mainly the expression of endogenic lectins, such as galectins and plant lectin, e.g. Dolichos biflorus agglutinin (DBA), and their glycoligands in the tear fluid, human corneal and conjunctival epithelium in physiological and disease conditions. Further, we studied the human natural antibody against Galα1,3Gal-R, which is mainly responsible for hyperacute rejection of xenografts transplants. We tried to investigate its localization in human corneal epithelium, lacrimal gland and tears. Material and Methods: Human tissue (lacrimal gland, tear fluid, conjunctiva, cornea, epidermis, keratinocyte and cultured corneal epithelium), as well as porcine tissue (cornea, liver and epidermis) were examined. Endogenous galectins (galectins-1, -3 and -7) were detected using immunohistochemistry methods. Binding sites for galectins, as well as binding sites for plant lectin Dolichos biflorus agglutinin, were localized by lectin histochemistry. Reverse lectin histochemistry was used for the study of binding reactivity of endogenous lectins using labelled (neo)glycoligands. Employing biotinylated natural human IgG anti -galactosides, as well as anti -galactosides, we detected reactive epitopes in human...
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Local production of cytokines after treatment with stem cells of damaged ocular surface
Kössl, Jan ; Holáň, Vladimír (advisor) ; Kovář, Marek (referee)
The damage of ocular surface represents one of the most common causes of decreased quality of vision or even blindness. If the injury is extensive and includes the region of limbus, niche of limbal stem cells (LSC), LSC deficiency occurs and the natural corneal regeneration is stopped. Conjunctival epithelium migrates into the injured area. Neovascularization, local inflammation and corneal opacity occur. Corneal transplantation is an insufficient treatment in such case. If the injury is bilateral, the allogenic limbal graft or LSC transplantation is required. In such cases systemic immunosuppressive drugs with many negative side-effects must be administered. The search for an adequate autologous substitution is important for avoid immunosuppressive medication. Mesenchymal stem cells (MSC) represent a perspective substitution for the reason of their immunomodulatory properties and the capability to differentiate in many cell types. There is possibility to isolate sufficient number of these cells from adipose tissue or bone marrow which are relatively easily accessible. Our goal was to observe local production of cytokines and other molecules which are present in inflammatory reaction after the chemical burn of the murine cornea and after the treatment with stem cells growing on nanofiber scaffold....
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