A Histomorphometric and Computational Investigation of the Stabilizing Role of Pectinate Ligaments in the Aqueous Outflow Pathway.

Murine models are commonly used to study glaucoma, the leading cause of irreversible blindness. Glaucoma is associated with elevated intraocular pressure (IOP), which is regulated by the tissues of the aqueous outflow pathway. In particular, pectinate ligaments (PLs) connect the iris and trabecular meshwork (TM) at the anterior chamber angle, with an unknown role in maintenance of the biomechanical stability of the aqueous outflow pathway, thus motivating this study. We conducted histomorphometric analysis and optical coherence tomography-based finite element (FE) modeling on three cohorts of C57BL/6 mice: 'young' (2-6 months), 'middle-aged' (11-16 months), and 'elderly' (25-32 months). We evaluated the age-specific morphology of the outflow pathway tissues. Further, because of the known pressure-dependent Schlemm's canal (SC) narrowing, we assessed the dependence of the SC lumen area to varying IOPs in age-specific FE models over a physiological range of TM/PL stiffness values. We found age-dependent changes in morphology of outflow tissues; notably, the PLs were more developed in older mice compared to younger ones. In addition, FE modeling demonstrated that murine SC patency is highly dependent on the presence of PLs, and that increased IOP caused SC collapse only with sufficiently low TM/PL stiffness values. Moreover, the elderly model showed more susceptibility to SC collapse compared to the younger models. In conclusion, our study elucidated the previously unexplored role of PLs in the aqueous outflow pathway, indicating their function in supporting TM and SC under elevated IOP.

Data on differentially expressed proteins in rock inhibitor-treated human trabecular meshwork cells using SWATH-based proteomics.

Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors represent a novel class of anti-glaucoma drugs because of their ocular hypotensive effects. However, the underlying mechanisms responsible for lowering intraocular pressure (IOP) are not completely clear. The protein profile changes in primary human trabecular meshwork (TM) cells after two days treatment with a ROCK inhibitor were studied using label-free SWATH acquisition. These results provided significant data of key protein candidates underlying the effect of ROCK inhibitor. Using the sensitive label-free mass spectrometry approach with data-independent acquisition (SWATH-MS), we established a comprehensive TM proteome library. All raw data generated from IDA and SWATH acquisitions were uploaded and published in the Peptide Atlas public repository (http://www.peptideatlas.org/) for general release (Data ID PASS01254).

Age-related changes in eye morphology and aqueous humor dynamics in DBA/2J mice using contrast-enhanced ocular MRI.

PURPOSE: Here, we are testing the hypothesis that dynamic contrast enhanced MRI (DCE-MRI) is a useful approach for non-invasively evaluating age-related changes in aqueous humor outflow and its contribution to elevated intraocular pressure in the DBA/2J model of pigmentary glaucoma. METHODS: A rodent-specific 7 T MRI was used to assess eye anatomy (anterior chamber (AC) and vitreous body (VB) morphology, eye size, lens size) and aqueous humor dynamics (via intravenous administration of Gd-DTPA and Gd-BOPTA contrast agents) in C57BL/6 and DBA/2J mice at 3 and 9 months of age. RESULTS: Gd-MRI was used to demonstrate an anterior solute pathway into the mouse AC. Topical latanoprost treatment in C57BL/6J mice reduced Gd-BOPTA accumulation in the AC. Age-related increases in AC area, AC depth and eye size were observed in DBA/2J mice compared to C57BL/6J mice. The rate of Gd-DTPA accumulation and peak Gd-DTPA intensity was lowest in 9-month old DBA/2J mice compared to 3-month old DBA/2J mice and C57BL/6J mice at both ages. Leakage of Gd-DTPA posteriorly into the VB was also observed in 9-month old DBA/2J mice. CONCLUSIONS: These studies support the idea that age-related changes in aqueous humor outflow contribute to elevated intraocular pressure (IOP) in the DBA/2J model of pigmentary glaucoma. Gd-MRI is a valuable tool for better understanding of mechanisms and dynamics of aqueous humor circulation in normal and glaucomatous mouse eyes or following topical administration of medicines to reduce IOP.

Schlemm's canal endothelia, lymphatic, or blood vasculature?

In the human eye, the final barrier for aqueous humor to cross before returning to systemic circulation is the inner wall of Schlemm's canal. Unfortunately, the specific contribution of the inner wall to total outflow resistance in the conventional pathway is unknown in both normal and glaucomatous eyes. To better understand inner wall physiology, we contrasted it with 2 specialized continuous endothelia, initial lymphatic, and blood capillary endothelia. Specifically, we compare their developmental origin, morphology, junctional complexes, microenvironment, and physiologic responses to different biomechanical factors. Our evaluation concludes that the inner wall of Schlemm's canal is unique, sharing extraordinary characteristics with both types of specialized endothelia in addition to having distinctive features of its own.

Targeted gene transfer to Schlemm's canal by retroperfusion.

The goal of the present study was to specifically modify protein expression in the resistance-generating region of the conventional outflow pathway, namely the inner wall of Schlemm's canal (SC) and the juxtacanalicular region of the trabecular meshwork, in perfused human anterior segments. Anterior segments from human cadaveric eyes were prepared for organ culture using standard techniques and were perfused at constant flow while recording pressure. After reaching a stable outflow facility within physiological limits, forward perfusion was stopped and a fluid-tight fence encircling the limbus was installed and filled with media containing an adenovirus encoding the lacZ reporter gene (either 2 x 10(6) or 6 x 10(6)PFU/ml). With the limbus submerged, pressure inside the chamber was lowered to -1 mmHg to facilitate reverse perfusion of virus into SC ("retroperfusion"). After 30-60 min at zero pressure (with some mixing), forward perfusion was restarted and continued for 5-7 days, after which anterior segments were fixed and processed for visualization of lacZ activity. Retroperfusion of nine anterior segments with adenovirus encoding a reporter gene did not appreciably alter baseline outflow facility (0.27+/-0.05 versus 0.29+/-0.08 microl/min per mmHg post-retroperfusion). Gross examination of outflow tissues showed focal distribution of lacZ activity around the circumference of SC, presumably near collector channels. In segments that were sequentially tilted during retroperfusion, the distribution of lacZ activity appeared more uniform. Sagittal histological sections showed lacZ activity in all portions of the conventional drainage tract, particularly cells in the resistance-generating region. Taken together, the results demonstrate that candidate protein expression by cells in the resistance-generating region of the conventional drainage pathway can be specifically modified by retroperfusion of adenovirus and examined for effects on outflow facility.