In vivo, biomechanical stress plays an important role in tissue physiology and pathology, affecting cell and tissue behavior. Even though conventional outflow tissues in the eye are constantly exposed to dynamic changes in intraocular pressure (IOP), the effects of such biomechanical stressors on outflow tissue function have not been analyzed. In particular, changes in IOP with each heartbeat have been measured in human eyes approximating 2.7 mmHg/sec. The purpose of this dissertation is to determine the effect(s) of ocular pulse on conventional outflow tissue regulation and the effect that contractility plays in this mechanical stress-mediated response. The central hypothesis directing this research is that cyclic intraocular pulsations (i.e. ocular pulse) play a significant role in conventional outflow facility.In order to address our hypothesis we studied the effect of biomechanical stressors on conventional outflow physiology using three different strategies: (1) by comparing conventional outflow endothelial cells to blood and lymphatic capillary endothelia, we gained a better understanding of the effects of biomechanical stress on conventional outflow tissue physiology, (2) by modifying the anterior segment perfusion model, we were able to measure the effect of ocular pulse on conventional outflow facility, and (3) by exposing trabecular meshwork cell monolayers to cyclic biomechanical pressure oscillations in the presence of compounds known to affect trabecular meshwork contractility, we were able to analyze the effect of rho-kinase-mediated contractility on the ocular pulse-associated response.Perfused human and porcine anterior segments showed a significant ocular pulse-mediated decrease in outflow facility; in addition, perfused trabecular meshwork monolayers showed an increase in intra-chamber pressure when exposed to cyclic pressure oscillations. This effect was blocked by Y27632 inhibition of rho-kinase-mediated contraction.In conclusion, the work shown in this dissertation demonstrates for the first time that trabecular outflow tissues are capable of responding to a physiologically-relevant cyclic biomechanical stress. This response can be observed as an increase in outflow resistance that translates to lower baselines in outflow facility of anterior segments and lower hydraulic conductivity of trabecular meshwork monolayers. In addition, we concluded that the observed ocular pulse-mediated response of trabecular meshwork cells is regulated by rho-kinase-induced contractility.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. Elevated intraocular pressure (IOP), resulting from increased aqueous humor outflow resistance, is a major risk factor for the development and progression of POAG. Outflow resistance in the trabecular outflow pathway is mainly (50-75%) generated in the juxtacanalicular connective tissue (JCT), and partially (25-50%) in the portion distal to the inner wall of Schlemm’s canal. The details of how aqueous humor flows through these tissues and how resistance in these tissues is regulated are not fully understood in normal and POAG eyes. Aqueous humor outflow was shown to be “segmental”, with discontinuous active regions of aqueous humor filtration along the trabecular outflow pathway that can be labeled with perfused fluorescent tracers and measured as effective filtration area (EFA). In this study, we investigated the relationship between changes in EFA along the trabecular outflow pathway and outflow facility/IOP under two experimental conditions. The first experiment was designed to increase outflow facility by using netarsudil, a recently approved Rho kinase inhibitor class glaucoma medication, in normal human donor eyes. The second experiment was designed to increase IOP with topical steroid treatment for 5 weeks in mice. The purpose of this study is to verify whether EFA can be modulated by netarsudil or steroid treatment and to demonstrate the morphological changes that may be responsible for the changes of EFA. We analyzed EFA along the trabecular outflow pathway and found that elevated/reduced EFA correlated with increased outflow facility/IOP. Guided by EFA, we performed detailed morphological comparison between the active and inactive portions of aqueous humor filtration tissue to evaluate possible structural changes involved in EFA regulation. We found that increased EFA was associated with a loosened JCT structure and dilated episclearal veins, while decreased EFA was associated with a compacted JCT structure, increased deposition of curly collagen and/or fibrillary structure in the trabecular meshwork, and increased basement membrane continuity. Our data suggest that the netarsudil/steroid-induced morphological changes in the trabecular outflow pathway can result in either an increase or decrease in EFA, which in turn contributes to the regulation of outflow facility/IOP. / 2020-10-24T00:00:00Z

Orientadores: Mônica Barbosa de Melo, José Paulo Cabral de Vasconcellos / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-25T15:53:03Z (GMT). No. of bitstreams: 1 Oliveira_MarianaBorges_M.pdf: 6901699 bytes, checksum: b84b4ac92949fdb082625b01ad6c66a1 (MD5) Previous issue date: 2014 / Resumo: O glaucoma engloba um grupo de doenças que têm como característica comum a atrofia progressiva do disco óptico com alteração correspondente de campo visual, decorrente da perda de células ganglionares da retina. O aumento da pressão intraocular (PIO) é o principal fator de risco para o desenvolvimento do glaucoma, mas tem sido sugerida a existência de outros aspectos relevantes, tais como alterações no metabolismo do óxido nítrico, na regulação do fluxo vascular, sinais de estresse oxidativo e alterações no sistema imunológico. O glaucoma primário de ângulo aberto (GPAA), a forma mais prevalente entre os glaucomas, está associado a uma série de fatores de risco para sua instalação e desenvolvimento, além da PIO. Os demais fatores de risco são: idade (relação direta), raça (mais frequente e grave em indivíduos da raça negra), miopia e história familiar de glaucoma. A maioria dos casos de GPAA apresenta um padrão complexo de hereditariedade, em que variantes de suscetibilidade contribuem para a gravidade da doença. O objetivo deste estudo do tipo caso-controle foi avaliar o papel de polimorfismos nos genes da interleucina alfa (IL1A), interleucina beta (IL1B) e fator de necrose tumoral alfa (TNFA) em relação à suscetibilidade ao GPAA. Após avaliação oftalmológica, foram recrutados 214 indivíduos não relacionados portadores de GPAA. A análise molecular foi realizada por meio de técnicas de PCR e sequenciamento direto. Neste estudo observou-se que o alelo C do polimorfismo -31 C/T, presente na região promotora do gene IL1B, confere risco para o glaucoma (p=0,002). Este alelo está contido em um "TATA" box, influenciando na transcrição deste gene. Existe um desequilíbrio de ligação entre os SNPs -31 C/T e -511 T/C do gene IL1B e este último também está associado a um risco aumentado para o glaucoma (p=0,006). Com relação aos demais SNPs estudados (+3954 C/T do gene IL1B, -889 C/T e +4845 G/T do gene IL1A e -238 G/A e -308 G/A do gene TNFA), não houve associação com o glaucoma. Este estudo sugere que há relação das variantes "C" do SNP -31 C/T e "T" do SNP -511 T/C do gene IL1B com a etiologia do GPAA em uma amostra da população brasileira / Abstract: Glaucoma comprises a group of diseases characterized by progressive atrophy of the optic disc and corresponding visual field loss, as consequence of retinal ganglion cells death. Augmented intraocular pressure (IOP) is the main risk factor for glaucoma development, but it has been suggested that there are other relevant aspects that may lead to neuronal and trabecular meshwork (TM) damage and contribute to the development of glaucoma, including alterations in nitric oxide metabolism, oxidative stress and vascular and immune system dysregulations. Primary open angle glaucoma (POAG), the most prevalent form of glaucoma, is associated with several risk factors to its initiation and progression besides IOP. The additional risk factors are: age (direct correlation), race (more frequent in black subjects), myopia and family history of glaucoma. The majority of POAG cases presents a complex pattern of heritability, in which susceptibility variants contribute to disease severity. The aim of this case-control study was to evaluate the role of single nucleotide polymorphisms (SNPs) in the interleukin alpha (IL1A), interleukin beta (IL1B) and tumor necrosis factor alpha (TNFA) genes in relation to POAG susceptibility. After ophthalmologic evaluation, 214 unrelated subjects with POAG were recruited. The molecular analysis was performed by PCR and direct sequencing techniques. In this study it was observed that the C allele of the -31 C/T promoter polymorphism of the IL1B gene confers risk for glaucoma (p=0.002). This allele disrupts a "TATA" box, influencing the transcription of the IL1B gene. There is linkage disequilibrium between the -31 C/T e -511 T/C SNPs, and the latter is also associated with an increased risk for glaucoma (p=0.006). Regarding the others studied SNPs (+3954 C/T of the IL1B gene, -889 C/T and +4845 G/T of the IL1A gene and -238 G/A and -308 G/A of the TNFA gene), there is no association with glaucoma. This study suggests the relationship of the "C" variant of -31C/T and "T" variant of -511T/C of IL1B gene with POAG etiology in a sample of the Brazilian population / Mestrado / Oftalmologia / Mestra em Ciências Médicas

Primary open-angle glaucoma (POAG) is a leading cause of blindness in the world. A primary risk factor for POAG is elevated intraocular pressure (IOP), caused by increased aqueous humor outflow resistance. Currently, lowering the IOP is the only effective way of treating glaucoma; however, the cause of increased outflow resistance remains unclear. This thesis will present a series of studies which investigated structures of the trabecular outflow pathway, including Schlemm’s canal endothelium, juxtacanalicular tissue, and trabecular beams, and their roles in regulating aqueous outflow resistance. The studies were conducted in both human and animal models using ex vivo ocular perfusion as well as in vitro microfluidic systems. In the first study, we investigated the effects of Y27632, a derivative of Rho-kinase inhibitor that is being developed as next generation glaucoma drug with unclear IOP lowering mechanism, on aqueous humor outflow dynamics and associated morphological changes in normal human eyes and laser-induced ocular hypertensive monkey eyes. In the second study, we developed and validated a novel three-dimensional microfluidic system using lymphatic microvascular endothelial cells. The microfluidic system can be used to study Schlemm’s canal endothelial cell dynamics and aqueous humor transport mechanism in the future. In the last study, we characterized the morphological structure, distribution, and thickness of the endothelial glycocalyx in the aqueous humor outflow pathway of human and bovine eyes. Together these studies will help define new directions for therapy that will help control IOP and preserve vision throughout a normal life span.

Primary open-angle glaucoma (POAG) is one of the leading causes of blindness worldwide. The only modifiable risk factor for POAG is elevated intraocular pressure, resulting from increased aqueous humor production or decreased drainage. Resistance to drainage in the aqueous outflow pathway is believed to reside in the juxtacanalicular connective tissue (JCT) and to be modulated by the inner wall (IW) endothelium of Schlemm’s canal (SC); however, the mechanisms that increase resistance in POAG remain unclear. To cross the IW, aqueous humor passes through I-pores on giant vacuoles (GVs) or B-pores between adjacent endothelial cells. Additionally, outflow around the circumference of the eye is segmental, or non-uniform, and fluorescent tracers can be used to label areas of high-flow and non-flow. The morphological differences in the endothelial cells of SC and their GVs in high- vs. non-flow areas have not been fully elucidated. In this project, we investigated the role of GVs and pores in the IW endothelial cells of SC in regulating segmental outflow in human eyes. We used serial block-face scanning electron microscopy to generate thousands of serial images and visualize these structures in 3D at the ultrastructural level. First, we 3D-reconstructed 45 individual IW cells and their GVs and quantified the number of connections each cell makes with the underlying JCT matrix/cells. We found that cells in high-flow areas made significantly fewer connections to JCT matrix/cells compared to cells in non-flow areas. Secondly, we analyzed 3,302 GVs for I-pores and basal openings and found a significantly greater percentage of GVs with both basal openings and I-pores in high-flow area compared to non-flow area, suggesting this type of GVs form a channel through which aqueous humor passes from JCT to SC. We also found that GVs with I-pores were significantly larger than those without I-pores. Our results suggest that decreasing number of cellular connections and increasing number of GVs with pores may be potential strategies to increase the amount of high-flow area and aqueous outflow for glaucoma treatment. Together, these studies add to our understanding of the role of GVs and pores in regulating segmental flow around the eye.

No description available.

Durch Ausübung physiologischer Grundfunktionen spielen Ionenkanäle eine entscheidende Rolle für die reguläre Funktion von Zellen. Zum besseren Verständnis ihrer Struktur und Funktion sind Untersuchungen natürlicher und künstlicher Ionenkanäle wichtige Werkzeuge. Großes analytisches und therapeutisches Potential ist in der Untersuchung künstlicher Kanäle in lebenden Zellen vorhanden, was bisher wenig Beachtung fand. In der vorliegenden Arbeit wurde die Wirkung der künstlichen Ionenkanäle THF-gram, THF-gram-TBDPS sowie linked-gram-TBDPS auf elektrophysiologische Eigenschaften boviner Trabekelwerkszellen des Auges anhand von Patch-Clamp-Untersuchungen im Whole-Cell-Modus analysiert. Die Untersuchung brachte folgende Erkenntnisse: 1. Die Inkorporation aller drei Verbindungen war erfolgreich, was sich durch Anstieg der Stromdichte und Verschiebung des Umkehrpotentials zeigte. 2. Einbau von THF-gram und THF-gram-TBDPS war mit dem Überleben der Zellen vereinbar, während linked-gram-TBDPS aufgrund einer sehr potenten Antwort bereits bei sehr geringen Konzentrationen zum raschen Zelltod führte. 3. Eine Asymmetrie der Stromantwort zugunsten stärkerer Auswärtsströme wurde bei THF-gram und in schwächerer Ausprägung bei THF-gram-TBDPS festgestellt. Linked-gram-TBDPS zeigte keine derartige Asymmetrie. 4. Unter Verwendung von Cs+ als Ladungsträger war der beobachtete Anstieg der Stromdichte bei allen drei Verbindungen eindeutig stärker als unter physiologischen Bedingungen (Na+/K+). 5. Die dargestellten Erkenntnisse sind ein erster Schritt zur therapeutischen Anwendung von künstlichen Ionenkanälen. Eine Weiterentwicklung in Richtung höherer Selektivität und besserer Kontrolle ist jedoch genauso erforderlich wie die Klärung der klinischen Umsetzbarkeit. / Ion channels play a pivotal role for regular cell function. To better understand their structure and function, investigation of both natural and artificial ion channels is being performed to date. Investigation of artificial channels in living cells hides a big potential, however, little attention has been paid to this field so far. In this work, the effect of the artificial ion channels THF-gram, THF-gram-TBDPS and linked-gram-TBDPS on electrophysiological properties of bovine trabecular meshwork cells was investigated with the patch-clamp-technique. Following results were obtained: 1. Incorporation of all three compounds was successful, which was proven by increase of current density and cell depolarisation. 2. The cells survived after incorporation of THF-gram and THF-gram-TBDPS but not after linked-gram-TBDPS, which resulted in cell death at very low concentrations. 3. Larger outward currents were observed with THF-gram and, at a lower extent, with THF-gram-TBDPS. Linked-gram-TBDPS did not show such an asymmetry. 4. With Cs+ as charge carrier all three compunds showed a stronger increase of current density than under physiological conditions (Na+/K+). 5. The decribed results are a first step towards therapeutic application of artificial ion channels, however, further development towards higher selectivity and better control is as necessary as clarification of clinical feasibility.

In open angle glaucoma, dysfunction of the trabecular meshwork (TM) results in impaired aqueous humour outflow leading to an elevated intraocular pressure (IOP) that underlies optic nerve damage and irreversible blindness. Currently, no curative treatment is available for the disease. Indeed, most pharmacological and surgical interventions usually provide only temporary relief from elevated IOP while little progress has been made in targeting the root cause of this disease: correcting the dysfunctional TM. In this context, we hypothesized that regeneration/refunctionalization of the TM may represent an effective therapeutic option to halt disease progression or even reverse the pathologic process. We previously demonstrated in a rat model of glaucoma that the injection of mesenchymal stromal cells (MSCs) cultured under hypoxic conditions or their conditioned media (MSC-CM) into laser-damaged TM area results in tissue regeneration. Injection of MSC or conditioned media in our glaucoma model led to activation and proliferation of ocular progenitor cells culminating in TM regeneration and a decrease in IOP. However, the mechanistic basis for this regenerative process remained elusive. Thus, the aim of this thesis is to elucidate the mechanistic basis of MSC secretome-mediated TM regeneration and the subsequent decrease in IOP. We now demonstrate that injection of hypoxic MSC-CM into laser-induced glaucomatous eyes resulted in massive immune cell recruitment. We also demonstrate that these hypoxic MSC-CM conditioned cells produced pro-regenerative factors in vitro and in vivo. Next, employing a proteomic approach, we identified and verified the pro-regenerative effect of several factors secreted by hypoxic MSC-CM recruited cells, which in turn induced the activation/proliferation of ocular progenitor cells leading to TM regeneration and decreased IOP. Upon individual injection of the purified factors into glaucomatous rat eyes, we observed a partial and delayed but significant decrease in IOP that correlated with an increase in the activation and proliferation of neuronal progenitor cells in the TM area. The co-injection of these factors resulted in a significant decrease in IOP compared with individual factor injection. Importantly, this drop in IOP was associated with restoration of retinal functionality, thus demonstrating the importance of these factors in the TM regeneration process and disease control. The findings presented in this thesis provide a novel acellular therapeutic approach for glaucoma treatment via in situ TM regeneration. Moreover, the knowledge gained here could have a lasting impact on how we induce tissue regeneration in other degenerative diseases and lead to novel therapeutic advances in regenerative medicine. / Dans le glaucome à angle ouvert, le dysfonctionnement du trabéculum (TM), un tissu nécessaire à la filtration de l'humeur aqueuse, entraîne une élévation de la pression intraoculaire (PIO). Ceci cause des lésions au niveau du nerf optique et une cécité irréversible. Présentement, aucun traitement curatif n'a été développé pour cette maladie. Nous émettons l'hypothèse que la régénération et re-fonctionnalisation du trabéculum peut représenter une option thérapeutique efficace pour arrêter ou inverser la progression de la maladie dans de nombreux cas de glaucome. Nous avons précédemment démontré les effets régénérateurs des cellules mésenchymateuses (MSCs) et de leurs milieux conditionnés par l'hypoxie (MSC-CM) dans la régénération du TM suite à un dommage par laser. Ce processus a conduit à l'activation et à la prolifération des cellules progénitrices oculaires résultant en une diminution de la PIO dans un modèle de glaucome induit par laser chez le rat. Cependant, la base mécanistique de ce processus de régénération reste encore inconnue. Ainsi, le but de cette thèse de recherche est d'élucider cette base mécanistique de la régénération du TM médiée par le sécrétome des MSC et la diminution subséquente de la PIO. À cette fin, l'injection de MSC-CM hypoxique dans les yeux glaucomateux induits par laser a entraîné un important recrutement de cellules immunitaires. Sous l’action du MSC-CM, ces cellules produisent des facteurs pro-régénératifs in vitro et in vivo. Ensuite, nous avons utilisé une approche protéomique et vérifié l'effet pro-régénératif des facteurs sécrétés par ces cellules exposées au MSC-CM hypoxique, sur l'activation et la prolifération des cellules progénitrices oculaires et la PIO. Lors de l'injection de ces facteurs chez le rat glaucomateux, nous avons observé une augmentation significative de l'activation et de la prolifération des cellules progénitrices neuronales présentes dans la zone du TM, résultant en une diminution de la PIO. De plus, l’injection combinée de ces facteurs résulte en une diminution synergique importante de la PIO. Cette baisse de la PIO était associée à une restauration de la fonction rétinienne, démontrant ainsi l'importance de ces facteurs dans le processus de régénération du TM et de contrôle de la maladie. Les résultats présentés dans cette thèse pourraient amener à une nouvelle approche thérapeutique acellulaire pour le traitement du glaucome via la régénération du TM. De plus, les connaissances acquises au cours de cette thèse pourraient avoir un impact durable sur la manière d’aborder la régénération tissulaire dans d'autres maladies dégénératives et amener des avancées thérapeutiques nouvelles en médecine régénératrice