The bulk of aqueous humour outflow resistance is generated in or near the inner wall endothelium of Schlemm's canal in normal eyes, and probably also in glaucomatous eyes. Fluid flow through this region is controlled by the location of the giant vacuoles and pores found in cells of the endothelium of Schlemm's canal, but the flow resistance itself is more likely generated either in the extracellular matrix of the juxtacanalicular connective tissue or the basement membrane of Schlemm's canal. Future studies utilizing in vitro perfusion studies of inner wall endothelial cells may give insights into the process by which vacuoles and pores form in this unique endothelium and why inner wall pore density is greatly reduced in glaucoma. Keywordsglaucoma; Schlemm's canal; hydraulic conductivity It has been recognized for more that 130 years that the elevated pressure characteristic of primary open-angle glaucoma arises due to an increased resistance to the outflow of aqueous humour from the eye. (Leber, 1873) However, a conclusive determination of where in the outflow pathways this elevated outflow resistance is generated has been elusive. Surprisingly, the locus of aqueous humour outflow resistance in the normal eye has also been not been unequivocally determined.Seidel (1921) using light microscopy, stated 'that the inner wall of Schlemm's canal stand in open communication with the anterior chamber, and that the aqueous humour directly washes around the inner wall endothelium of Schlemm's canal and is only separated from the lumen of Schlemm's canal by a thin, outer membrane'. Our view is little different today. The locus of outflow resistance, both in the normal eye and the glaucomatous eye, is thought to arise either in the endothelial lining of Schlemm's canal, or very near to this location. In this article, current thoughts on where that flow resistance might be generated are reviewed.There are a number of excellent review articles (Tripathi, 1974a,b;Bill, 1975;Bill and Mäepea, 1994;Gong et al., 1996;Johnson and Erickson, 2000;Ethier, 2002) that describe the detailed morphology and physiology of the aqueous outflow pathway. In this review, the evidence that leads to the conclusion that the inner wall region is responsible for the bulk of aqueous humour outflow resistance is first presented. Then, attention is focused on those proximal aspects of this pathway that are nearest to the endothelial linings of Schlemm's canal, and the transport characteristics of these structures.The bulk of the aqueous humour flows out of the anterior chamber of the eye through the conventional aqueous outflow pathway comprised of the trabecular meshwork, the juxtacanalicular connective tissue, the endothelial lining of Schlemm's canal, Schlemm's canal itself, the collecting channels and aqueous veins, and then finally drains into the episcleral NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript venous system, rejoining the blood from whence it came (in this review, the juxtacanalicular...

Aqueous humor outflow resistance is the primary determinant of intraocular pressure (IOP), and increased outflow resistance is the basis for elevated IOP associated with glaucoma. Experimental evidence suggests that the bulk of outflow resistance is generated in the vicinity of the inner wall endothelium of Schlemm's canal, its basement membrane and the juxtacanalicular connective tissue (JCT). However, attempts to sort out the contribution of each of these tissues to total outflow resistance have not been successful.Conventional understanding of outflow resistance assumes that the resistance of each tissue strata (i.e., the inner wall endothelium, its basement membrane and JCT) in the outflow pathway adds in series to contribute to total outflow resistance generation. However, this perspective leads to a paradox where the apparent resistances of all tissues in the outflow pathway are much lower than the measured total resistance. To resolve this paradox, we explore synergistic models of outflow resistance generation where hydrodynamic interactions between different tissue strata lead to a total resistance that is greater than the sum of the individual tissue resistances. We closely examine the "funneling" hypothesis that has emerged as a leading synergistic model, and we review the basis of funneling, mechanical and biological requirements for funneling and evidence in support of this hypothesis. We also propose refinements to the funneling model and describe how funneling may relate to segmental variability of aqueous humor outflow patterns observed within the trabecular meshwork.Pressure gradients across the JCT and inner wall endothelium will generate mechanical loads that influence the morphology of these tissues. Because tissue morphology may in turn affect outflow resistance, there exists the potential for a two-way coupling or a "fluid-solid interaction" between outflow hydrodynamics and the mechanical behavior of the inner wall and JCT. Furthermore, the adhesions and tethers between the inner wall and JCT must be physically capable of supporting such loads. We examine the structure and mechanical strength of these adhesions, and provide evidence that these adhesions and tethers are unable to support the full load imposed by the bulk of outflow resistance generation unless a substantial fraction of outflow resistance is generated within the JCT, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. consistent with the funneling model. This indicates that these attachments between the inner wall and JCT have considerable physiological importance for outflow resis...

We examined whether the presence of the cell cortex might explain, in part, why previous studies using atomic force microscopy (AFM) to measure cell modulus (E) gave higher values with sharp tips than for larger spherical tips. We confirmed these AFM findings in human umbilical vein endothelial cells (HUVEC) and Schlemm's canal (SC) endothelial cells with AFM indentation ≤ 400 nm, two cell types with prominent cortices (312 ± 65 nm in HUVEC and 371 ± 91 nm in SC cells). With spherical tips, E (kPa) was 0.71 ± 0.16 in HUVEC and 0.94 ± 0.06 in SC cells. Much higher values of E were measured using sharp tips: 3.23 ± 0.54 in HUVEC and 6.67 ± 1.07 in SC cells. Previous explanations for this difference such as strain hardening or a substrate effect were shown to be inconsistent with our measurements. Finite element modeling studies showed that a stiff cell cortex could explain the results. In both cell types, Latrunculin-A greatly reduced E for sharp and rounded tips, and also reduced the ratio of the values measured with a sharp tip as compared to a rounded tip. Our results suggest that the cell cortex increases the apparent endothelial cell modulus considerably when measured using a sharp AFM tip.

Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm's canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell-likely accounting for increased outflow resistance-that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness-and its biomechanical effects on pore formation-as a therapeutic target in glaucoma.cell mechanics | primary open-angle glaucoma | modulus | cytoskeleton

SummaryPersistent free-running circannual (approximately year-long) rhythms have evolved in animals to regulate hormone cycles, drive metabolic rhythms (including hibernation), and time annual reproduction. Recent studies have defined the photoperiodic input to this rhythm, wherein melatonin acts on thyrotroph cells of the pituitary pars tuberalis (PT), leading to seasonal changes in the control of thyroid hormone metabolism in the hypothalamus. However, seasonal rhythms persist in constant conditions in many species in the absence of a changing photoperiod signal, leading to the generation of circannual cycles. It is not known which cells, tissues, and pathways generate these remarkable long-term rhythmic processes. We show that individual PT thyrotrophs can be in one of two binary states reflecting either a long (EYA3+) or short (CHGA+) photoperiod, with the relative proportion in each state defining the phase of the circannual cycle. We also show that a morphogenic cycle driven by the PT leads to extensive re-modeling of the PT and hypothalamus over the circannual cycle. We propose that the PT may employ a recapitulated developmental pathway to drive changes in morphology of tissues and cells. Our data are consistent with the hypothesis that the circannual timer may reside within the PT thyrotroph and is encoded by a binary switch timing mechanism, which may regulate the generation of circannual neuroendocrine rhythms, leading to dynamic re-modeling of the hypothalamic interface. In summary, the PT-ventral hypothalamus now appears to be a prime structure involved in long-term rhythm generation.