Measurement of Outflow Facility Using iPerfusion

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.

Visualization of conventional outflow tissue responses to netarsudil in living mouse eyes

“…Using an iPerfusion system (Sherwood et al, 2016), we subjected enucleated mouse eyes to 9 sequential pressure steps (4.5-21 mmHg) and measured flow. A sample flow-pressure plot for a C57 mouse is given in figure 3A, showing a higher rate of outflow for a given pressure in the netarsudil treated eye.…”

“…A system especially designed to simultaneously measure low outflow facilities in paired mouse eyes, the iPerfusion system (Sherwood et al, 2016), was used in the present study. Each freshly enucleated mouse eye was mounted on a stabilization platform in a perfusion chamber using a small amount of cyanoacrylate glue (Loctite, Westlake Ohio, USA).…”

“…Data analysis was carried out as described previously (Sherwood et al, 2016). Briefly, a non-linear flow-pressure model was used to account for the pressure dependence of outflow facility in mice, and the reference facility was analyzed at a reference pressure of 8 mmHg (approximates the physiological pressure drop across the conventional outflow pathway in living mice).…”

“…Representative experiment showing flow rate (Q) vs pressure (P) that was used to calculate outflow facility in a single pair of eyes. Panel B. Cello plot (Sherwood et al, 2016) shows percentage change in facility for each pair of eyes in response to treatment with netarsudil. Dark blue error bars indicate confidence interval from the fitting of the model to the flow-pressure data and lighter error bars include additional uncertainty arising from differences between contralateral eyes of C57BL/6 mice (Sherwood et al, 2016).…”

Open-angle glaucoma: therapeutically targeting the extracellular matrix of the conventional outflow pathway

“…As ECM accumulates, the extracellular space close to SCEC becomes more tortuous, leading to increased outflow resistance [42]. The effect that this ECM has on outflow resistance alone is difficult to measure; however, recent developments in measuring and modeling of perfusion data may lead to more accurate estimations of outflow facility [78].…”