Measurement of Outflow Facility and Ocular Biomechanical Properties

Previous work has shown that the mechanical properties of pelvic floor tissues are significantly affected by 8 weeks after OVX.19 Thus, we chose to house the animals for 8 weeks after surgery before euthanizing the animals using CO₂. Eyes were then enucleated and we measured aqueous outflow facility and ocular biomechanical properties using the iPerfusion system,20 a custom-designed combination of hardware and software that simultaneously records pressure and fluid flow entering the eye during ocular perfusion. Although the system was originally designed to assess outflow facility,20 it is also capable of measuring ocular compliance.21 All perfusion experiments were begun within 30 minutes of enucleating the eyes.

On each day of testing, the pressure and flow sensors in the iPerfusion system were calibrated, and the system compliance was measured. The system compliance was later used to determine ocular compliance by subtracting the system compliance from the total compliance (compliance of the system plus that of the eye). Enucleated eyes were secured on a stage in a heated (37°C) PBS bath, and the anterior chamber was cannulated with a 33-gauge (G) needle (NanoFil; World Precision Instruments, Sarasota, FL, USA). The eye was perfused with filtered Dulbecco's PBS with 5.5-mM glucose (DBG) and equilibrated at 13 mm Hg until a steady state was reached, typically requiring between 30 and 45 minutes. We then perfused the eye at eight equally divided pressure steps from 12 to 36 mmHg (Fig. 1), ensuring that a steady state was reached at each step based on a previously established threshold.20 We calculated the instantaneous ratio of flow/pressure, then evaluated the slope against time over a moving window of 450 seconds. When the slope was less than 0.1 nl/min/mm Hg/min continuously for a minute, steady state was considered to have been reached.

Ocular perfusion protocol. (Left) Representative pressure versus time trace during a perfusion. After an acclimatization period of 30 minutes at 13 mm Hg, the pressure is reduced to 12 mm Hg, at which point the perfusion is considered to begin (t = 0). Pressure is then increased in eight steps. The center panel illustrates the corresponding inflow versus time. (Right) Overview of the discrete volume method used to determine ocular volume at each pressure increment. The blue shaded region represents aqueous humor outflow, and the overlying brown shaded region indicates the flow involved in increasing the ocular volume. For each pressure step, the area of this region indicates the total volume increase (system plus eye), from which ocular compliance can be calculated.

In agreement with previous studies in mice,20^,22 our preliminary tests confirmed that the Brown Norway rat exhibits zero flow at zero pressure; therefore, we fit the outflow behavior to a power law as previously described:20

where P is intraocular pressure, C_r is the outflow facility at a reference pressure P_r,c, and β is a fitting parameter characterizing the pressure-dependent change in facility. We chose a reference pressure (P_r,c) of 15 mm Hg (Fig. 2) as a representative physiological pressure drop across the conventional outflow pathway for Brown Norway rats, which have a typical in vivo resting IOP of 20 mm Hg and for which we assumed an episcleral venous pressure of 5 mm Hg.23

Representative flow, outflow facility, and ocular compliance versus pressure behavior in Brown Norway rats. Flow (left), outflow facility (center), and ocular compliance (right) are functions of pressure. The orange symbols represent measured values at each pressure step, the dashed orange lines are the corresponding fits, and the solid black lines represent the 95% confidence intervals of each fit. The vertical orange lines indicate the reference pressures (15 mm Hg and 20 mm Hg) at which outflow facility (C_r) and ocular compliance (ϕ_r) were determined.

To assess ocular tissue stiffness, we measured ocular compliance (ϕ), the incremental change in ocular volume per incremental change in IOP, corrected for system compliance as noted above.21 A more compliant eye implies that the corneoscleral shell is less stiff; thus, ocular compliance is an indirect measure of corneoscleral connective tissue properties. The compliance–pressure relationship can be described by a modified Friedenwald equation:

where ϕ_r is the ocular compliance at a reference pressure P_r,ϕ, which we chose to be 20 mm Hg (Fig. 2), corresponding to the average IOP of the Brown Norway rat.23 The parameter γ is an empirical parameter that accounts for deviation of the compliance–pressure relationship from the Friedenwald model (for which γ = 0).

Ocular compliance was calculated following the previously described discrete volume method,21 in which the compliance is defined as the change in ocular volume for a given change in pressure, ϕ = ∆V/∆P. For a given pressure step j, the ocular compliance can be calculated as

where ∆P_j is the change in pressure between two steps, T is the time at the end of the step, Q is the flow rate (nl/min) measured by the flow sensor, C is the outflow facility (see Equation 1), and ϕ_s is the system compliance. The term (Q – CP) describes the flow rate of fluid that is accumulating within the eye (i.e., which is expanding the corneoscleral shell); hence, the integral in Equation 3 provides the volume change of the eye (∆V_j) in response to the change in pressure (∆P_j).

As the ocular compliance itself changes during a pressure step, the calculated ocular compliance ϕ|Pϕ,j corresponds to an intraocular pressure (P_ϕ,j) that lies between the limits of the pressure for step j and is determined using the mean value theorem:21

Equation 2 can be fit to the acquired P_ϕ,j and ϕ|Pϕ,j values to calculate ϕ_r and γ. However, as γ is not known a priori, an iterative process is applied, which is described elsewhere,21 by initially assuming that γ = 0 to calculate P_ϕ,j values with Equation 4, then using these to estimate γ using Equation 2 and repeating.