**Purpose.**:
Mice are commonly used in glaucoma research, but relatively little is known about aqueous outflow dynamics in the species. To facilitate future use of the mouse as a model of aqueous humor outflow, several fundamental physiological parameters were measured in the mouse eye.

**Methods.**:
Eyes from adult mice of either sex (C57BL/6 background) were enucleated, cannulated with a 33-gauge needle, and perfused at constant pressure while inflow was continuously measured.

**Results.**:
At 8 mm Hg, total outflow facility (*C* _{total}) was 0.022 ± 0.005 μL/min/mm Hg (all values mean ± SD; *n* = 21). The flow–pressure relationship was linear up to 35 mm Hg. The conventional outflow facility (*C* _{conv}) was 0.0066 ± 0.0009 μL/min/mm Hg, and the unconventional outflow (*F* _{u}) was 0.114 ± 0.019 μL/min, both measured at room temperature. At 8 mm Hg, 66% of the outflow was via the unconventional pathway. In a more than 2-hour-long perfusion at 8 mm Hg, the rate of facility change was 2.4% ± 5.4% (*n* = 11) of starting facility per hour. The ocular compliance (0.086 ± 0.017 μL/mm Hg; *n* = 5) was comparable to the compliance of the perfusion system (0.100 ± 0.004 μL/mm Hg).

**Conclusions.**:
Mouse eyes are similar to human eyes, in that they have no detectable washout rate and a linear pressure–flow relationship over a broad range of intraocular pressures. Because of the absence of washout and the apparent presence of a true Schlemm's canal, the mouse is a useful model for studying the physiology of the inner wall of Schlemm's canal and the conventional outflow tissues.

^{ 1 }), and shallow angle, all of which conspire to make studying aqueous humor drainage in the mouse technically challenging.

^{ 2 }using cannulation and two-level outflow facility determination, were the first to characterize aqueous humor dynamics in mouse eyes. Subsequently, Aihara et al.,

^{ 3 }Zhang et al.

^{ 4 }and Millar et al. (

*IOVS*2008;49:ARVO E-Abstract 354) also perfused mouse eyes by using a two-level approach, with either pressure or flow control. The two-level approach is the gold standard for in vivo facility measurements, but it is difficult to perform certain manipulations in vivo (e.g., very long-term perfusions and looking at a wide range of pressures).

^{ 5 }Briefly, it consisted of a computer-controlled syringe pump that delivered a variable flow rate (

*Q*) to the anterior chamber so as to maintain a desired IOP, as monitored by the pressure transducer connected to a computer control system (Labview software; National Instruments, Newbury, UK). The syringe pump (model 33; Harvard Apparatus, Holliston, MA) had a specially machined lead screw (40 threads/in.), which allowed higher accuracy at lower flow rates with less oscillation.

*IOVS*2008;49:ARVO E-Abstract 354).

^{ 3,6 }Flow and pressure were measured at 10 Hz and electronically recorded every 10 seconds. Total outflow facility (

*C*

_{total}) was calculated as

*C*

_{total}= total inflow rate (

*F*)/intraocular pressure (IOP), where we assume that at equilibrium, the total inflow rate equals the total outflow rate.

*C*

_{0}) was determined as the first stable total facility reading after cannulation. A linear regression analysis was performed on the facility trace to determine the slope (

*m*). Washout rate was defined as

*m*divided by

*C*

_{0}(i.e., the percentage change in total facility per hour). To exclude the possibility of slow particulate obstruction of the outflow pathways, in some experiments we prefiltered the perfusion medium through a 0.45-μm and then a 0.22-μm syringe filter (Sartorius Stedim Biotech, Göttingen, Germany), just before perfusion.

*n*= 3). This resistance was negligible compared with the outflow resistance of a mouse eye and could therefore be ignored during interpretation of the perfusion results. The compliance of the perfusion system was measured to be 0.100 ± 0.004 μL/mm Hg. The ocular compliance was measured to be 0.086 ± 0.017 μL/mm Hg (

*n*= 5 eyes, Fig. 1). Note that the ocular compliance was comparable to the overall system compliance.

*C*

_{total}= 0.022 ± 0.005 μL/min/mm Hg (mean ± SD,

*n*= 21; Fig. 2). At other perfusion pressures, total outflow facilities were 0.034 ± 0.006 μL/mm Hg/min at 4 mm Hg (

*n*= 8) and 0.014 ± 0.004 μL/mm Hg/min at 15 mm Hg (

*n*= 13). Although we did not look at a wide range of mouse ages, we did not see any clear relationship between total outflow facility and mouse age. Figure 3 is an example of the results from a perfusion experiment at three different pressure levels (regimen 2).

^{ 7 }At pressures greater than 35 mm Hg, flow rate increased more rapidly as pressure was increased, implying an increasing total facility at higher pressures, the opposite of the situation in human eyes.

^{ 7 }It is also noteworthy that when IOP was returned to 15 mm Hg at the end of the experiment, the measured flow rate agreed almost perfectly with that measured earlier in the perfusion at 15 mm Hg, demonstrating that the IOP elevation during the perfusion did not damage or otherwise irreversibly alter the outflow pathway tissues.

*C*

_{conv}) which was 0.0066 ± 0.0009 μL/min/mm Hg which would be the same as measured by two-level perfusion. This result is significantly different from the total outflow facility (at 8 mm Hg) which is the slope of the line A–B in Figure 4B and is equal to

*C*

_{total}= 0.022 ± 0.005 μL/min/mm Hg. The intercept of the regression line is the unconventional (pressure-independent) flow rate,

*F*

_{u}= 0.114 ± 0.019 μL/min. From the data in Figure 4B, it is possible to compute the percentage of outflow that is pressure independent (

*F*

_{u}/

*F*). It can be observed (Fig. 4C) that this percentage is maximum at low pressures and then declines at higher pressures. At 8 mm Hg (corresponding to approximately ∼15 mm Hg in vivo), 66% of outflow is pressure independent. These values are comparable to published data.

^{ 3 }

*n*= 11, Fig. 5A), not significantly different from 0, implying that (similar to human eyes

^{ 8 }) there was no detectable washout in the mouse eye. These results did not depend on whether the perfusate was prefiltered. Histologic examination showed that, after extended perfusions, there were no evident morphologic changes in the aqueous outflow tissues (Fig. 5B).

^{ 9 }the absence of washout as shown here further confirms that the mouse is a useful model for studying the physiology of the inner wall of Schlemm's canal and the conventional outflow tissues.

*C*

_{conv}= 0.0066 μL/mm Hg/min and the unconventional outflow rate to be

*F*

_{u}= 0.114 μL/min. These values are similar to published data obtained in vivo (Millar JC, et al.

*IOVS*2008;49:ARVO E-Abstract 354),

^{ 2 –4,10 }(e.g., conventional outflow facilities in the range of 0.005 to 0.0146 μL/min/mm Hg and

*F*

_{u}in the range of 0.113 to 0.148 μL/min have been reported). This result suggests that the use of enucleated (postmortem) eyes did not appreciably alter aqueous drainage behavior.

^{ 11 }considered the effect of temperature on aqueous outflow in perfused human eyes and concluded that, so long as changes in the viscosity of the perfusion fluid with temperature were accounted for, outflow facility was approximately constant between 22°C and 37°C. If we assume that a similar conclusion holds for the mouse eye, then we can estimate the facility at 37°C from our data. The correction factor is the ratio of PBS viscosities between room temperature (on average, ∼22°C) and body temperature, namely 1.38,

^{ 12 }giving a conventional outflow facility of

*C*

_{conv}= 0.0091 μL/mm Hg/min and an unconventional outflow rate of

*F*

_{u}= 0.157 μL/min. These values are consistent with previous measurements.

^{ 13 }: where

*F*is total outflow rate,

*F*

_{u}is the pressure-independent (unconventional) outflow rate, EVP is episcleral venous pressure, and

*C*

_{conv}is the conventional outflow facility. When perfusing enucleated eyes, we and others have frequently computed “the facility,” defined as the ratio of flow,

*F*, to IOP, which we have referred to as the total facility,

*C*

_{total}, in this work. Equation 1 shows that when unconventional outflow,

*F*

_{u}, is negligible and EVP is 0 (as is the case in an enucleated eye), then

*C*

_{conv}and

*C*

_{total}are the same. However, this is clearly not the case for the mouse eye, where

*F*

_{u}is non-negligible so that

*C*

_{conv}≠

*C*

_{total}, even for an enucleated eye. This conclusion is expressed graphically in Figure 4B, where

*C*

_{conv}and

*F*

_{u}are the slope and intercept of the regression line (solid line) respectively, and

*C*

_{total}is the slope of the dashed line AB.

*C*

_{conv}and

*F*

_{u}, respectively, as expressed in equation 1. The fact that our derived values were similar to those determined in the living eye by two-level perfusion indicates that pressure-independent outflow must not have been grossly affected by the eye being enucleated, where EVP is 0 and blood flow to the eye is absent.

^{ 5 }For example, bovine and monkey eyes both show washout rates of approximately 20% per hour during first 2 hours of perfusion with buffered saline solutions.

^{ 14 –17 }In the mouse eye, over the course of a 2- to 3-hour perfusion at a constant pressure of 8 mm Hg, we observed less than a 3% facility increase. This finding may be due in part to the high unconventional outflow in the mouse eye, which would suggest that only a fraction of the infused perfusion fluid exits the eye through the trabecular meshwork, thus reducing the effects on the meshwork and inner wall. Alternatively, it may be that mouse eyes do wash out, but at a rate that is so small to be undetectable with present capabilities, or simply that the mouse eye does not wash out.

^{ 8 }We therefore expect that this approach will be valid in mouse eyes as well, so long as the eyes are freshly obtained and not damaged during enucleation. Another limitation was that we could not completely avoid anterior chamber deepening. However, in some eyes, the needle tip contacted the iris and even created an iridotomy allowing communication between the posterior and anterior chambers. In such eyes, we did not observe any effect on facility. This apparent lack of an effect of anterior chamber deepening on facility in mice may be due to the relatively large lens in the mouse eye, which supports the iris and likely prevents significant posterior iris displacement compared with eyes of other species.

^{ 18 }reported that unconventional outflow represents 20.5% of total outflow in BALB/cJ mice. This is 3-fold smaller than the 66% unconventional outflow in C57BL/6 mice reported here and smaller than the ∼80% unconventional outflow reported by other groups.

^{ 3,4,10 }The nature of these differences is uncertain, but may reflect strain-dependent differences in uveoscleral outflow or scleral permeability or methodological differences associated with enucleation (e.g., removal of extraocular tissues, evaporation of medium from the ocular surface, or elimination of EVP). Because of the importance of uveoscleral outflow and its potentially large role in the mouse eye, these differences in unconventional outflow between strains warrant further investigation.

*P*

_{e}(with respect to atmosphere) and

*V*

_{e}, respectively. Because the eye is filled with an incompressible fluid, conservation of mass requires that where

*t*is time and

*F*

_{in}and

*F*

_{out}are the inflow and outflow rates for the eye, respectively. Because the eye is enucleated, there is no aqueous humor production and

*F*

_{in}is provided entirely by flow through the needle. Allowing the eye to be modeled as an elastic shell with a constant compliance, β

_{e}leads to where

*V*

_{eo}is the volume of the enucleated eye at 0 IOP. When the EVP is 0, as is the case in an enucleated eye, the Goldmann equation can be written as where

*F*

_{u}is pressure-independent (unconventional) outflow and

*R*

_{e}is the conventional (pressure-dependent) outflow resistance, both of which are assumed to be constant. Note that

*R*

_{e}= 1/

*C*

_{conv}, as defined in Figure 4B.

*F*

_{in}: The behavior of

*F*

_{in}, however, depends on whether the perfusion is configured to be constant pressure or constant flow rate. Importantly, because of system compliance,

*F*

_{in}is not trivially determined, even in the case of constant flow rate perfusion. Next, we describe the formulation for

*F*

_{in}for the constant pressure and constant flow rate perfusion cases.

^{ 5 }). Because each control system has its own intrinsic response time, we consider here the optimal case of a constant-height perfusion reservoir.

*R*

_{n}and tubing with compliance β

_{t}connected to the reservoir. We assume that the resistance of the tubing is negligible compared to the needle, such that the filling time of the tubing is very short and that

*P*, the pressure upstream of the needle, is a constant determined by the height of the reservoir. The pressure drop across the needle is therefore

_{n}*P*

_{n}−

*P*

_{e}, and the flow rate through the needle is equal to

*F*

_{in}: Combining equations 5 and 6 yields the governing equation describing the time response of

*P*

_{e}for constant-pressure perfusion, Because

*R*

_{e}≫

*R*

_{n}, equation 7 can be approximated as where it is apparent that the characteristic time constant for

*P*

_{e}to reach steady state is equal to

*R*

_{n}β

_{e}. Equation 8 was solved numerically for relevant parameter values (NDSolve function in Mathematica; Wolfram, Champaign, IL) to produce the dashed curve in Figure 6.

*F*

_{o}to the upstream end of the compliant tubing. If the resistance of the tubing is much lower than the needle resistance, then the pressure is approximately uniform throughout the tubing, with a value equal to

*P*

_{n}. However, in this case,

*P*

_{n}is no longer a constant, but is dictated by the tubing compliance β

_{t}according to where

*V*

_{t}is the volume of the tubing and

*V*

_{to}is the tubing volume at

*P*

_{n}= 0. Because the contents of the tubing are incompressible, we may write where

*F*

_{in}is the flow rate into the eye through the needle as described by equation 6. Combining equations 5, 6, 9, and 10 yields: The numerical solution of equation 11, using parameters relevant for the mouse eye and our perfusion system is shown by the solid curve in Figure 6.