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.

Osteoarthritis is the most common form of arthritis. Genetic models have been developed to determine if and how a targeted gene may influence cartilage degenerative changes. The itga1-null mouse model has an inhibited integrin α1B1 through a ubiquitous integrin α1 subunit knockout, which leads to fibrosis in articular cartilage through excessive signalling from transforming growth factor beta (TGFB). Depleting this TGFB signalling is proposed to have a protective effect on cartilage. This project is part of a foregoing study where a cartilage-specific knockout of TGFB receptor type II (TBRII) was used to deplete TGFB signalling in articular cartilage of the itga1-null mice to reduce the severity of cartilage degradation. This project continues the analysis of the genetic model into bone architecture at the knee. Mouse hindlimbs were scanned at a 13μm resolution using micro-computed tomography and segmented into 3D datasets containing calcified tissues and bone of the knee and surroundings. Quantification methods for trabecular bone parameters (bone volume fraction, trabecular separation, and trabecular thickness) and ectopic calcification of soft tissues were developed. Loss of trabecular bone around the involved joint is a hallmark of post-traumatic osteoarthritis. However, the results from this study showed no significant changes in trabecular bone of itga1-null mouse knees despite observing severe osteoarthritic changes in the adjacent cartilage. There were no significant effects in peri-articular trabecular bone when the TBRII knockout in cartilage was activated, but there were significant increases in ectopic calcifications of the menisci and collateral ligaments. These ectopic calcifications were also seen in tamoxifen control mice, suggesting that tamoxifen, along with TBRII depletion in cartilage, had a role in increased abnormal calcifications. Although integrin α1B1 inhibition appears to have an important role in cartilage degeneration, it does not appear to influence the bony changes that normally accompany post-traumatic arthritis. / Thesis / Master of Applied Science (MASc) / Osteoarthritis is a common joint disorder, associated mainly with cartilage degradation. Some genes have been identified that cause or prevent osteoarthritis. A previous study used two of these genes in a genetic mouse model to explore how osteoarthritis may develop. Removing the integrin α1 subunit from mice caused osteoarthritic changes in the cartilage of the mouse knee. When the transforming growth factor beta gene was removed from the cartilage, these changes were less severe. This project continued the study by exploring changes in bone around the mouse knee. We quantified bone changes around the mouse knee using high-resolution micro-computed tomography scans. Contrary to common findings in post-traumatic osteoarthritis, we found that there were no significant changes in the bone around the knees even where severe cartilage changes had been identified. However, there were significant increases in calcifications of soft tissues including the meniscus and ligaments around the knee.

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The "Knuckle-walking Hominin Hypothesis" postulates that there was a knuckle-walking phase during the transition from quadrupedalism to bipedalism. To address this question, previous research has focused on the search for a "signal" within the wrist, and metacarpals of extant knuckle walkers that can be used to infer this locomotor pattern in extinct hominins. To date, the examined features have not yielded a clear, non-contested signal. I explore the Knuckle-walking Hominin Hypothesis in two ways: 1. by examining the hand postures and the manual pressure application of Pan and Gorilla during knuckle walking to determine whether there are species specific differences and 2. by examining the internal and external morphology of the manual phalanges in an attempt to isolate a clear "knuckle-walking signal". Chimpanzees are more variable in their preferred contact digits, and use both hand positions with equal frequency ("palm-in" - palm facing toward the body and "palm-back" - palm facing posteriorly). In contrast, gorillas consistently make contact with all four digits 2-5, maintain a pronated arm, and use the palm-back hand position. In both taxa, hand position affects which digit acts as the final touch-off element and therefore receives maximum pressure in a given step, and digit 5 receives significantly less pressure than the other rays. Gorillas are, in effect, practicing a refined subset of the variety of knuckle-walking postures used by the more arboreal chimpanzees. A clear knuckle-walking signal is seen in both the external and internal morphology of the phalanges. Chimpanzees and gorillas have the same middle phalangeal curvature profile with the greatest curvature found in digit 5 (5 > 2 > 3 > 4), the element that receives the least amount of pressure. This phalangeal curvature profile is a feature not shared with any of the included taxa practicing different modes of locomotion. They also have similar Indices of Relative Curvature (IRC-middle phalangeal curvature/proximal phalangeal curvature) for digits 2-5 that clearly delineate them with "flatter" middle phalanges and more curved proximal phalanges (IRCs = ~0.85), from quadrupeds with more curved middle than proximal phalanges (IRCs > 1), and suspensory primates with higher and more equal curvature values for both elements (IRCs = ~1). This ability to differentiate between locomotor groups holds if the IRCs are composed of elements from different rays of the same manus and from elements of different individuals. Within the trabecular bone structure, knuckle walkers are differentiated from quadrupeds and suspsensory primates in 3 locations: the metacarpal head, and the proximal ends of the middle and proximal phalanges. In particular, the metacarpal head shows distinct differences between the groups: knuckle walkers have a palmar-dorsal alignment of trabeculae and disc-like shape, suspensory taxa have a proximodistal alignment and rod-like shape and quadrupeds have a proximodistal alignment and disc-like shape. The ability to differentiate between locomotor categories using isolated zones increases the applicability of these signals to a fragmentary and limited fossil record. The morphological similarities, specifically the shared curvature profile, and the similar knuckle-walking kinematics employed by chimpanzees and gorillas point to a shared origin of knuckle walking.

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Yes / We analyzed the trabecular bone changes in the pubic bone (PB) and in the auricular surface (AS) of the ilium using 319 CT scans of cadavers to estimate the age. Although the sharpness of the trabecular structure decreases in CT images when soft tissues are present, we identified four phases for the changes in PB and five in AS; a juvenile trait in PB and a senile trait in AS helped narrow the age range. High correlation with age was identified for both sexes in PB (F 0.89; M 0.75) and in AS (F 0.85; M 0.71) used independently or combined (F 0.91; M 0.78). The old adults (>60 years) could be evaluated with better accuracy and discriminated in several phases. We found low inter-observer error and low inaccuracy (about 6 years, mean for all age ranges). The method is robust with respect to slice thickness, display window and kernel within the tested ranges.