Abstract
Elevated intraocular pressure (IOP) is the predominant risk factor for glaucoma, and reducing IOP is the only successful strategy to prevent further glaucomatous vision loss. IOP is determined by the balance between the rates of aqueous humour secretion and outflow, and a pathological reduction in the hydraulic conductance of outflow, known as outflow facility, is responsible for IOP elevation in glaucoma. Mouse models are often used to investigate the mechanisms controlling outflow facility, but the diminutive size of the mouse eye makes measurement of outflow technically challenging. In this study, we present a new approach to measure and analyse outflow facility using iPerfusion™, which incorporates an actuated pressure reservoir, thermal flow sensor, differential pressure measurement and an automated computerised interface. In enucleated eyes from C57BL/6J mice, the flow-pressure relationship is highly non-linear and is well represented by an empirical power law model that describes the pressure dependence of outflow facility. At zero pressure, the measured flow is indistinguishable from zero, confirming the absence of any significant pressure independent flow in enucleated eyes. Comparison with the commonly used 2-parameter linear outflow model reveals that inappropriate application of a linear fit to a non-linear flow-pressure relationship introduces considerable errors in the estimation of outflow facility and leads to the false impression of pressure-independent outflow. Data from a population of enucleated eyes from C57BL/6J mice show that outflow facility is best described by a lognormal distribution, with 6-fold variability between individuals, but with relatively tight correlation of facility between fellow eyes. iPerfusion represents a platform technology to accurately and robustly characterise the flow-pressure relationship in enucleated mouse eyes for the purpose of glaucoma research and with minor modifications, may be applied in vivo to mice, as well as to eyes from other species or different biofluidic systems.
Highlights
Intraocular pressure (IOP) is regulated by the balance between the secretion of aqueous humour (AH) and its outflow across the hydrodynamic resistance of the conventional outflow pathway
We described iPerfusion, a constant pressure perfusion system that provides faster and more accurate measurements of outflow facility in enucleated mouse eyes, relative to perfusion systems previously described in the literature
We developed a statistical analysis and the weighted t-test to account for uncertainties in the perfusion measurements and in the estimation of facility, and devised graphical formats to present the data in a manner that appropriately displays the uncertainty and spread in the measurements
Summary
Intraocular pressure (IOP) is regulated by the balance between the secretion of aqueous humour (AH) and its outflow across the hydrodynamic resistance of the conventional outflow pathway. The considerable disadvantage of mouse eyes is their diminutive size, being approximately 3% of the volume of AH compared to human eyes [21, 22], which corresponds to extremely low flow rates, on the order of 50 nl/min at physiological IOP This makes the assessment of outflow facility extremely sensitive to uncertainties in the methods of measurement and data analysis. Fitting a straight line to the data to estimate facility is only appropriate if C is independent of pressure and Q0 has a finite value The validity of these assumptions in the context of enucleated mouse eyes will be investigated in the present study. Additional sources of error in these methods include estimation of the height of the reservoir, non-linearity in the pressure sensor or weight measurement, inaccuracies in specification of D (as the flow rate scales with D2) and evaporation.
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