all experiments were performed in compliance with the association for research in vision and ophthalmology ( arvo ) statement for the use of animals in ophthalmic and vision research and the approval of the jackson laboratory institutional animal care and use committee ( iacuc ) . all mice ( 35 months old and matched sexes )
the mice were kept on a standard 14-hour light/10-hour dark cycle with food and water available ad libitum .
a mouse eye perfusion apparatus was engineered based on a human anterior segment perfusion apparatus ( fig . 1 , supplementary fig .
our apparatus , milled from lexan , consisted of a base with a conical frustum for mounting eyes ( fig . 1 , supplementary fig . s1).the top of the frustum had a diameter of 2.7 mm and the base 3.4 mm .
we built our first system based on the human apparatus , which had a large footprint ( supplementary fig .
we then redesigned that base to reduce the footprint and for ease of manufacture ( fig .
1 ) . most importantly the frustum design and performance were identical in both devices ( supplementary fig .
the frustum had fluid inlet and outlet channels running from the base to the top . in our newer systems , 26-gauge needles ( becton dickinson , franklin lakes , nj , usa )
were inserted into the inlet and outlet channels at the frustum base and bent gently to a curve .
the needles were sealed into the inlet and outlet channels ( henceforth ports ) using epoxy glue .
( a ) the device consists of a base with central frustum onto which the eye is mounted .
the frustum has two fluid ports ( see [ b ] ) for addition and removal of fluids .
the eye is held in place using an o - ring that seals the eye against the frustum .
a mounting washer and 4 screws hold the o - ring in place creating a tight seal .
( c ) the mounted eye is oriented so that approximately 45% of the limbal tissue , including the drainage structures , is clear of where the o - ring clamps the tissue .
( see supplementary methods ) was kept moist , and mounted carefully and gently onto the frustum using the opening in the side of the globe .
the flaps cut into the sclera allowed the eye to sit on the frustum , and the flaps were lightly glued onto the platform bearing the frustum ( using tissue glue ) to hold the eye in position .
a seal was created between the eye and frustum using an o - ring and washer ( fig .
the washer was secured in place by four corner screws that insert into the base . for uniformity ,
all screws were symmetrically and equally tightened using a torque screwdriver ( 10 cn.m ; fig .
the limbus of the eye spans the top of the frustum with sclera on one side and cornea on the other side .
although the retina is removed , the retinal pigment epithelium layer is maintained intact and lines the sclera .
the eye was checked for a pressure - tight seal by gently pushing in test perfusate ( dulbecco 's pbs [ dpbs ] , balanced salt solution [ bss ] , or a drug mixture ) through the inlet port , after closing the outlet port , using a three - way stopcock to determine if the flaccid eye mounted on the frustum inflated without visible leaks around the o - ring . the inlet port then was connected to a pressure transducer ( dtxplus tnf - r ; argon medical systems , plano , tx , usa ; see supplementary methods for details ) , in series with a perfusate - filled 100 l nanofil syringe ( worcester polytechnic institute [ wpi ] , worcester , ma , usa ) attached to a ump3 pump ( wpi ) controlled by a micro-4 pump controller ( wpi ) .
the transducer was connected to the apparatus with a short length of pressure - resistant tubing .
any air bubbles in the system were removed carefully using the perfusion fluid of choice .
the pressure transducer connects to the computer via a digitizer ( measurement computing , norton , ma , usa ) and with the microprocessor controller we created closed - loop control ( wpi ; see below ) .
the eye was maintained in a moist state at 37c by inverting the eye over a well of a heating block filled with water ( to a height approximately 1 mm below the eye , see fig .
( a ) the eye is sealed onto the perfusion device and fluid ( saline , drugs , and tracers ) is introduced or drained through the two ports ( f - in , f - out ) .
the f - in port is connected to a pressure transducer , which in turn is connected to a micropump .
the pressure transducer monitors iop constantly and supplies this information to the computer , which control the pump controller and pump .
the control algorithm maintains pressures at the desired values and durations according to the preset experimental plan .
( b ) schematic showing a mounted eye in a humidifying chamber that is maintained at 37c ( not to scale ) .
while measuring outflow , fluid was perfused at a controlled rate to maintain the eye at a specific pressure .
the perfusion rate was monitored and controlled by an in - house computer program ( labview ; national instruments , austin , tx , usa ) using a bang - bang control algorithm .
the steady state error of the system was 0.5 mm hg . once started , the program continuously monitored and recorded information from the pressure transducer and flow rate information from the syringe pump microprocessor controller .
if pressure fell by a small fraction of 1 mm hg below the set level , the program triggered the syringe pump to perfuse fluid into the eye via the microprocessor controller .
as soon as the pressure increased to a fraction of 1 mm hg above the set point the pump turned off . as the pump delivered fluid in increments of 0.67 nl / step , flow continued after the pump was shut off , explaining the oscillatory nature of the flow rate plot .
the software was designed to set different experimental pressure levels in a stepwise manner ( up to six pressure values can be input ) .
the user set the  rise  time to reach a set pressure , the  equilibration  time for the eye to adjust to that pressure , and a  record  time to measure flow rate required to maintain the pressure .
the rise and equilibration time ensured that we recorded data only after the flow rate oscillations had reached a stable state . before outflow measurement on any day , we calibrated the pressure transducer using a mercury manometer
. outflow was measured by obtaining the flow rate required to maintain a series of set pressures .
we use two pressure series : protocol a  5 , 15 , 25 , 35 , 45 , and then 15 mm hg again , and protocol b  8 , 12 , 16 , 20 , 24 , and 12 mm hg again . in both protocols ,
rise time was 5 minutes , equilibration time was 5 minutes , and record time was 10 minutes ( see monitoring and control section above ) .
a run was considered successful if the following criteria were met in analysis of the data : ( 1 ) flow rates at each pressure achieved stable oscillation about a mean with oscillations being no more than 30 nl for 5 , 15 , and 25 mm hg pressure set points , and ( 2 ) flow rates were similar at the first and second 15 mm hg ( protocol a ) or first and second 12 mm hg ( protocol b ) .
if an eye failed at any pressure , all data for that eye were discarded .
drugs were perfused in at 15 mm hg for 30 minutes followed by 15 minutes at 5 mm hg before measuring outflow .
we used y27632 at 200 m and l - n - nitroarginine methyl ester ( l - name ) at 100 m .
no eyes were perfused for longer than 2 hours and some eyes were fixed for immunofluorescence or histology ( see supplementary methods ) after outflow measurements were concluded .
all experiments were performed in compliance with the association for research in vision and ophthalmology ( arvo ) statement for the use of animals in ophthalmic and vision research and the approval of the jackson laboratory institutional animal care and use committee ( iacuc ) . all mice ( 35 months old and matched sexes )
the mice were kept on a standard 14-hour light/10-hour dark cycle with food and water available ad libitum .
a mouse eye perfusion apparatus was engineered based on a human anterior segment perfusion apparatus ( fig . 1 , supplementary fig .
s1 ) . our apparatus , milled from lexan , consisted of a base with a conical frustum for mounting eyes ( fig . 1 , supplementary fig . s1).the top of the frustum had a diameter of 2.7 mm and the base 3.4 mm .
we built our first system based on the human apparatus , which had a large footprint ( supplementary fig .
we then redesigned that base to reduce the footprint and for ease of manufacture ( fig .
1 ) . most importantly the frustum design and performance were identical in both devices ( supplementary fig .
the frustum had fluid inlet and outlet channels running from the base to the top . in our newer systems ,
26-gauge needles ( becton dickinson , franklin lakes , nj , usa ) were inserted into the inlet and outlet channels at the frustum base and bent gently to a curve .
the needles were sealed into the inlet and outlet channels ( henceforth ports ) using epoxy glue .
( a ) the device consists of a base with central frustum onto which the eye is mounted .
the frustum has two fluid ports ( see [ b ] ) for addition and removal of fluids .
the eye is held in place using an o - ring that seals the eye against the frustum .
a mounting washer and 4 screws hold the o - ring in place creating a tight seal .
( c ) the mounted eye is oriented so that approximately 45% of the limbal tissue , including the drainage structures , is clear of where the o - ring clamps the tissue .
a dissected eye ( see supplementary methods ) was kept moist , and mounted carefully and gently onto the frustum using the opening in the side of the globe .
the flaps cut into the sclera allowed the eye to sit on the frustum , and the flaps were lightly glued onto the platform bearing the frustum ( using tissue glue ) to hold the eye in position .
a seal was created between the eye and frustum using an o - ring and washer ( fig .
the washer was secured in place by four corner screws that insert into the base . for uniformity ,
all screws were symmetrically and equally tightened using a torque screwdriver ( 10 cn.m ; fig .
the limbus of the eye spans the top of the frustum with sclera on one side and cornea on the other side .
although the retina is removed , the retinal pigment epithelium layer is maintained intact and lines the sclera .
the eye was checked for a pressure - tight seal by gently pushing in test perfusate ( dulbecco 's pbs [ dpbs ] , balanced salt solution [ bss ] , or a drug mixture ) through the inlet port , after closing the outlet port , using a three - way stopcock to determine if the flaccid eye mounted on the frustum inflated without visible leaks around the o - ring . the inlet port then was connected to a pressure transducer ( dtxplus tnf - r ; argon medical systems , plano , tx , usa ; see supplementary methods for details ) , in series with a perfusate - filled 100 l nanofil syringe ( worcester polytechnic institute [ wpi ] , worcester , ma , usa ) attached to a ump3 pump ( wpi ) controlled by a micro-4 pump controller ( wpi ) .
the transducer was connected to the apparatus with a short length of pressure - resistant tubing .
any air bubbles in the system were removed carefully using the perfusion fluid of choice .
the pressure transducer connects to the computer via a digitizer ( measurement computing , norton , ma , usa ) and with the microprocessor controller we created closed - loop control ( wpi ; see below ) .
the eye was maintained in a moist state at 37c by inverting the eye over a well of a heating block filled with water ( to a height approximately 1 mm below the eye , see fig .
( a ) the eye is sealed onto the perfusion device and fluid ( saline , drugs , and tracers ) is introduced or drained through the two ports ( f - in , f - out ) .
the f - in port is connected to a pressure transducer , which in turn is connected to a micropump .
the pressure transducer monitors iop constantly and supplies this information to the computer , which control the pump controller and pump .
the control algorithm maintains pressures at the desired values and durations according to the preset experimental plan .
( b ) schematic showing a mounted eye in a humidifying chamber that is maintained at 37c ( not to scale ) .
while measuring outflow , fluid was perfused at a controlled rate to maintain the eye at a specific pressure . the perfusion rate was monitored and controlled by an in - house computer program ( labview ; national instruments , austin , tx , usa ) using a bang - bang control algorithm .
the steady state error of the system was 0.5 mm hg . once started , the program continuously monitored and recorded information from the pressure transducer and flow rate information from the syringe pump microprocessor controller .
if pressure fell by a small fraction of 1 mm hg below the set level , the program triggered the syringe pump to perfuse fluid into the eye via the microprocessor controller .
as soon as the pressure increased to a fraction of 1 mm hg above the set point the pump turned off .
as the pump delivered fluid in increments of 0.67 nl / step , flow continued after the pump was shut off , explaining the oscillatory nature of the flow rate plot . the software was designed to set different experimental pressure levels in a stepwise manner ( up to six pressure values can be input ) .
the user set the  rise  time to reach a set pressure , the  equilibration  time for the eye to adjust to that pressure , and a  record  time to measure flow rate required to maintain the pressure .
the rise and equilibration time ensured that we recorded data only after the flow rate oscillations had reached a stable state .
before outflow measurement on any day , we calibrated the pressure transducer using a mercury manometer . outflow was measured by obtaining the flow rate required to maintain a series of set pressures .
we use two pressure series : protocol a  5 , 15 , 25 , 35 , 45 , and then 15 mm hg again , and protocol b  8 , 12 , 16 , 20 , 24 , and 12 mm hg again . in both protocols ,
rise time was 5 minutes , equilibration time was 5 minutes , and record time was 10 minutes ( see monitoring and control section above ) .
a run was considered successful if the following criteria were met in analysis of the data : ( 1 ) flow rates at each pressure achieved stable oscillation about a mean with oscillations being no more than 30 nl for 5 , 15 , and 25 mm hg pressure set points , and ( 2 ) flow rates were similar at the first and second 15 mm hg ( protocol a ) or first and second 12 mm hg ( protocol b ) .
if an eye failed at any pressure , all data for that eye were discarded .
drugs were perfused in at 15 mm hg for 30 minutes followed by 15 minutes at 5 mm hg before measuring outflow .
we used y27632 at 200 m and l - n - nitroarginine methyl ester ( l - name ) at 100 m .
no eyes were perfused for longer than 2 hours and some eyes were fixed for immunofluorescence or histology ( see supplementary methods ) after outflow measurements were concluded .
the system consisted of a perfusion platform with a conical frustum for mounting the eye ( fig . 1 , supplementary fig .
we first determined the best way to mount eyes onto the frustum . due to mouse anatomy , mounting the anterior segment of the eye 
cornea up ,  as with human anterior segments , placed the limbus too close to all clamping mechanisms that we devised .
thus , we mounted the eye with the limbus up and spanning the top of the frustum ( therefore , 45% of the limbal circumference was available for perfusion ; see supplementary methods ) .
this approach provided ample clearance from the o - ring and an orientation that would ultimately facilitate live imaging . to enable this
, we developed the dissection scheme that is shown in supplementary figures s3 and s4 ( see supplementary methods ) .
the dissection of the eye is a critical and delicate step that requires attention to detail . before performing any outflow measurements , we tested the system for any inherent resistance and compliance ( defined as change in flow rate in response to pressure caused by any elasticity in the system ) .
it is critical to determine these parameters because they can make outflow measurement inaccurate . upon using a flow rate of 660 l / min ( far larger than the flow observed by an eye ) and leaving the inlet port open ( no eye secured to system )
, we observed no increase over atmospheric pressure ( 0 mm hg ) , indicating that the system had no measurable resistance at this flow rate ( or under physiologic conditions ) .
system compliance was determined to be 20.65 nl / mm hg ( see supplementary methods ) .
washout ( c increase upon prolonged ocular perfusion ) is observed in some nonhuman species , including primates but not humans or mice .
the washout rate for mouse eyes using our system was 2.5  1.96% ( n = 6 ) , in agreement with an earlier study ( washout rate = 2.4% ) and in contrast to monkey eyes , where the washout rate is 26% to 42% .
thus , we confirmed that mouse eyes , like human eyes , have negligible washout .
we determined c in mouse eyes by computing the slope of the flow rate versus pressure plot ( protocols a and b , see supplementary methods ) .
importantly , this plot shows that the flow rates at 15 mm hg at the beginning and end of the perfusion protocol are similar , indicating that the drainage tissue is undamaged .
flow rates versus pressure graphs are depicted in figures 4a ( protocol a , n = 10 ) and 4b ( protocol b , n = 8) . in these graphs ,
the plotted curve approximated linearity up to 25 mm hg and showed an upward deviation after 25 mm hg ( fig .
the linear nature is clear when outflow is measured using more points within the narrower pressure range of protocol b. protocol a was used to understand flow rate changes over a wide range of pressure .
protocol b , with more points in the linear range , will be useful when there is a need for high precision measurement of suspected flow rate changes at low pressures or when changes are subtle .
the plot shows the flow rate and pressure traces recorded during perfusion of a representative eye ( protocol a , see methods ) .
the axis on the left shows flow rate ( nl / min ) and the one on the right pressure ( mm hg ) .
( a ) eyes ( n = 10 ) perfused at 5 , 15 , 25 , 35 , and 45 mm hg ( protocol a ) .
the flow rate to pressure relationship is biphasic : it is linear up to 25 mm hg ( dashed line ) , after which its rate of change increased .
the equation of the line for the linear range was y = 0.0047x + 0.0175 , and the quality of linear fit r = 0.99946 .
the slope of flow rate change with pressure in this linear range is the c = 4.7  1.2 nl / min / mm hg .
when corrected to match whole eyes ( as only 45% of drainage tissue is available in mounted eyes ) c = 10.4  2.64 nl / min / mm hg .
( b ) eyes ( n = 8) perfused over a narrower pressure range of 8 , 12 , 16 , 20 , and 24 mm hg ( protocol b ) .
the line of fit for the linear part of the plot , y = 0.004x + 0.03 , r = 0.9995 , gives a c value of 4.0  0.6 nl / min / mm hg .
when corrected for the whole eye c = 8  1.32 nl / min / mm hg .
we obtained c values of 4.7  1.2 nl / min / mm hg ( mean  sd ) using protocol a and 4.0  0.6 nl / min / mm hg by protocol b in eyes of b6 mice .
there was no significant difference in c values obtained by either protocols a or b ( p = 0.095 ) .
the y - intercepts in the flow rate versus pressure plots , often considered a measure of pressure - independent flow , was 17.5 nl / min and 30 nl / min for protocols a and b , respectively . the absolute value of facility depends on how much of the outflow pathway is being perfused . in our experiments , approximately 45% of the limbal circumference ( see supplementary methods ) was available for perfusion .
therefore , the measured facility must be increased by a factor of 2.2 to estimate the facility for the entire outflow pathway .
thus , the c values for the whole eye would be 10.34  2.64 nl / min / mm hg using protocol a and 8.0  1.32 nl / min / mm hg using protocol b. to determine if the drainage structures are intact after outflow assessment , we used our recently described technique and prox1-gfp eyes to image large swaths of the sc at a subcellular level after outflow measurement ( as sc inner wall is the last barrier to drainage ) . following protocol a ( n = 6 , fig .
5 ) , vascular endothelial ( ve)cadherin staining showed that the cell shape and cell junctions were intact and comparable in control and perfused eyes ( fig .
retention of green fluorescent protein ( gfp ) signal in sc after outflow , and lack of intracellular propidium iodide ( included in perfusate , data not shown ) in limbal tissue , indicated the absence of membrane damage to the drainage structure cells .
these results also indicated that the integrity of sc inner wall endothelia was uncompromised at a cellular level .
similarly , conventional sections ( see supplementary methods ) showed that a perfused eye ( protocol a ) had an open sc and intact tm that were comparable to those in a control eye with intact iris ( fig .
5c ) . using a fluorescent 3-kda dextran tracer , we also showed that flow was restricted to the drainage structures , sc and tm .
( a ) whole mount of a prox1-gfp eye after an outflow experiment , showing the entire extent of the limbus and sc ( green ) subjected to flow .
cadherin staining ( not shown ) was present in area low in prox1-gfp at the right .
arrowheads indicate points where the o - ring contacted the limbus when the eye was mounted on the frustum . typically approximately 45% of the limbus is subjected to flow ( see supplementary methods ) .
( b ) higher magnification images of a freshly dissected control eye and an eye after outflow .
the general morphology ( gfp , green ) and cellular junctions ( ve - cadherin , red ) of sc are indistinguishable between these eyes .
the gfp level in eyes subjected to outflow is always within the range of variability in unperfused control prox1-gfp eyes .
retention of gfp signal after outflow is a good indicator that sc cells are healthy .
( it is well established that compromised cell membrane integrity results in leakage of cytoplasmic gfp and reduction in green fluorescence . )
scales bars : ( a ) 300 m ; ( b ) top 100 m ; insets 30 m .
( c ) plastic sections of a control ( 1 ) and perfused ( 2 ) eye showing the drainage structures .
n = 3 eyes each for control and outflow cohorts . images at lower magnification show the angle in the context of the iris and cornea ( top ) .
higher magnification images ( bottom ) allow for visualization of the open sc and tm .
we next determined if perturbation of outflow at a molecular level can be detected using our perfusion device .
we first tested the effect of the rock inhibitor , y-27632 ( see supplementary methods for details ) , on c. y-27632 is well established to increase outflow in other species .
we observed a 74.4% increase in c in eyes treated with y-27632 ( n = 10 , t - test p = 0.003 ; fig . 6 ) .
thus , as expected , rock inhibition increased c. this experiment validated the use of our system to measure changes in c. rho - associated protein kinase inhibitor y27632 increases outflow in mouse eyes .
eyes treated with y27632 were perfused with 200 m drug in bss for 30 minutes at 15 mm hg , followed by a 15 minutes of perfusion at 5 mm hg before measuring outflow ( protocol a ) .
the lines of fit for the linear part of the plots are : control , y = 0.0047x + 0.0175 , r = 0.99946 ; y27632 , y = 0.0082x + 0.0003 , r = 0.9996 .
c was different between groups : control , c = 4.7  1.2 nl / min / mm hg ( whole eye c = 10.4  2.64 nl / min / mm hg ) ; y27632 , c = 8.2  3 nl / min / mm hg ( whole eye c= 18.04  6.6 nl / min / mm hg ) .
nos3 ( endothelial nitric oxide synthase , enos ) and cav1 are genes implicated in glaucoma .
we first disrupted nos3 function by inhibition using the inhibitor l - name ; see supplementary methods for details ) as well as through genetic mutation .
treatment with l - name caused a reduction in c by 28.3% from control ( n = 7 eyes ; t - test , p = 0.025 ; fig .
c was reduced by 35.8% compared to control ( n = 7 eyes ; t - test , p = 0.008 ; fig .
next , we performed outflow measurements on cav1 null ( cav1 ) eyes ( n = 9 eyes ; fig . 8) . in cav1 eyes ,
c was reduced by 47.8% compared to control ( t - test , p = 0.02 ) .
we detected this reduction in two independent experiments on a b6 strain background and once on a mixed strain background ( not shown ) .
the requirement of nos3 activity was tested using nos3 mutant or l - name  treated wild - type eyes .
( a ) flow - rate versus pressure graph obtained from l - name  treated and control eyes .
( b ) flow - rate versus pressure graph obtained from nos3 and control eyes .
black diamonds represent control eyes and black triangles either l - name  treated eyes or nos3 eyes .
the equations representing the trendline for the linear part of the curve ( in [ a ] and [ b ] ) were : control , y = 0.0053x + 0.0254 , r = 0.9997 ; l - name , y = 0.0038x + 0.0382 , r = 0.99971 ; and nos3 
 y = 0.0034x + 0.0211 , r = 0.9976 .
c was different between groups : control , c = 5.3  1.4 nl / min / mm hg ( whole eye c = 11.6  3.08 nl / min / mm hg ) ; l - name , c = 3.8  0.7 nl / min / mm hg ( whole eye c = 8.36  1.54 nl / min / mm hg ) ; nos3 , c = 3.4  0.6 nl / min / mm hg ( whole eye c = 7.48  1.32 nl / min / mm hg ) .
flow - rate versus pressure graph showing a comparison of flow rates in control and cav1 eyes .
the equations representing the trend line for the linear part of the curve were : control , y = 0.007x + 0.0326 , r = 0.99322 , black circles ; cav1 , y = 0.0036x + 0.039 , r = 0.99322 , black diamond .
control , c = 6.9  3.4 nl / min / mm hg ( whole eye c = 15.18  4.4 nl / min / mm hg ) , and c = 3.6  2.5 nl / min / mm hg ( whole eye c = 7.92  5.5 nl / min / mm hg ) .
the system consisted of a perfusion platform with a conical frustum for mounting the eye ( fig . 1 , supplementary fig .
we first determined the best way to mount eyes onto the frustum . due to mouse anatomy , mounting the anterior segment of the eye 
cornea up ,  as with human anterior segments , placed the limbus too close to all clamping mechanisms that we devised .
thus , we mounted the eye with the limbus up and spanning the top of the frustum ( therefore , 45% of the limbal circumference was available for perfusion ; see supplementary methods ) .
this approach provided ample clearance from the o - ring and an orientation that would ultimately facilitate live imaging . to enable this
, we developed the dissection scheme that is shown in supplementary figures s3 and s4 ( see supplementary methods ) .
the dissection of the eye is a critical and delicate step that requires attention to detail .
before performing any outflow measurements , we tested the system for any inherent resistance and compliance ( defined as change in flow rate in response to pressure caused by any elasticity in the system ) .
it is critical to determine these parameters because they can make outflow measurement inaccurate . upon using a flow rate of 660 l / min ( far larger than the flow observed by an eye ) and leaving the inlet port open ( no eye secured to system ) ,
we observed no increase over atmospheric pressure ( 0 mm hg ) , indicating that the system had no measurable resistance at this flow rate ( or under physiologic conditions ) . system compliance was determined to be 20.65 nl / mm hg ( see supplementary methods ) .
washout ( c increase upon prolonged ocular perfusion ) is observed in some nonhuman species , including primates but not humans or mice .
the washout rate for mouse eyes using our system was 2.5  1.96% ( n = 6 ) , in agreement with an earlier study ( washout rate = 2.4% ) and in contrast to monkey eyes , where the washout rate is 26% to 42% .
thus , we confirmed that mouse eyes , like human eyes , have negligible washout .
we determined c in mouse eyes by computing the slope of the flow rate versus pressure plot ( protocols a and b , see supplementary methods ) .
importantly , this plot shows that the flow rates at 15 mm hg at the beginning and end of the perfusion protocol are similar , indicating that the drainage tissue is undamaged .
flow rates versus pressure graphs are depicted in figures 4a ( protocol a , n = 10 ) and 4b ( protocol b , n = 8) .
in these graphs , the plotted curve approximated linearity up to 25 mm hg and showed an upward deviation after 25 mm hg ( fig .
the linear nature is clear when outflow is measured using more points within the narrower pressure range of protocol b. protocol a was used to understand flow rate changes over a wide range of pressure .
protocol b , with more points in the linear range , will be useful when there is a need for high precision measurement of suspected flow rate changes at low pressures or when changes are subtle .
the plot shows the flow rate and pressure traces recorded during perfusion of a representative eye ( protocol a , see methods ) .
the axis on the left shows flow rate ( nl / min ) and the one on the right pressure ( mm hg ) .
( a ) eyes ( n = 10 ) perfused at 5 , 15 , 25 , 35 , and 45 mm hg ( protocol a ) .
the flow rate to pressure relationship is biphasic : it is linear up to 25 mm hg ( dashed line ) , after which its rate of change increased .
the equation of the line for the linear range was y = 0.0047x + 0.0175 , and the quality of linear fit r = 0.99946 .
the slope of flow rate change with pressure in this linear range is the c = 4.7  1.2 nl / min / mm hg .
when corrected to match whole eyes ( as only 45% of drainage tissue is available in mounted eyes ) c = 10.4  2.64 nl / min / mm hg .
( b ) eyes ( n = 8) perfused over a narrower pressure range of 8 , 12 , 16 , 20 , and 24 mm hg ( protocol b ) .
the line of fit for the linear part of the plot , y = 0.004x + 0.03 , r = 0.9995 , gives a c value of 4.0  0.6 nl / min / mm hg .
when corrected for the whole eye c = 8  1.32 nl / min / mm hg .
we obtained c values of 4.7  1.2 nl / min / mm hg ( mean  sd ) using protocol a and 4.0  0.6 nl / min / mm hg by protocol b in eyes of b6 mice .
there was no significant difference in c values obtained by either protocols a or b ( p = 0.095 ) .
the y - intercepts in the flow rate versus pressure plots , often considered a measure of pressure - independent flow , was 17.5 nl / min and 30 nl / min for protocols a and b , respectively . the absolute value of facility depends on how much of the outflow pathway is being perfused . in our experiments , approximately 45% of the limbal circumference ( see supplementary methods ) was available for perfusion .
therefore , the measured facility must be increased by a factor of 2.2 to estimate the facility for the entire outflow pathway .
thus , the c values for the whole eye would be 10.34  2.64 nl / min / mm hg using protocol a and 8.0  1.32 nl / min / mm hg using protocol b. to determine if the drainage structures are intact after outflow assessment , we used our recently described technique and prox1-gfp eyes to image large swaths of the sc at a subcellular level after outflow measurement ( as sc inner wall is the last barrier to drainage ) . following protocol a ( n = 6 , fig .
5 ) , vascular endothelial ( ve)cadherin staining showed that the cell shape and cell junctions were intact and comparable in control and perfused eyes ( fig .
retention of green fluorescent protein ( gfp ) signal in sc after outflow , and lack of intracellular propidium iodide ( included in perfusate , data not shown ) in limbal tissue , indicated the absence of membrane damage to the drainage structure cells .
these results also indicated that the integrity of sc inner wall endothelia was uncompromised at a cellular level .
similarly , conventional sections ( see supplementary methods ) showed that a perfused eye ( protocol a ) had an open sc and intact tm that were comparable to those in a control eye with intact iris ( fig .
5c ) . using a fluorescent 3-kda dextran tracer , we also showed that flow was restricted to the drainage structures , sc and tm .
( a ) whole mount of a prox1-gfp eye after an outflow experiment , showing the entire extent of the limbus and sc ( green ) subjected to flow .
cadherin staining ( not shown ) was present in area low in prox1-gfp at the right .
arrowheads indicate points where the o - ring contacted the limbus when the eye was mounted on the frustum . typically approximately 45% of the limbus is subjected to flow ( see supplementary methods ) .
( b ) higher magnification images of a freshly dissected control eye and an eye after outflow .
the general morphology ( gfp , green ) and cellular junctions ( ve - cadherin , red ) of sc are indistinguishable between these eyes .
the gfp level in eyes subjected to outflow is always within the range of variability in unperfused control prox1-gfp eyes .
retention of gfp signal after outflow is a good indicator that sc cells are healthy .
( it is well established that compromised cell membrane integrity results in leakage of cytoplasmic gfp and reduction in green fluorescence . )
scales bars : ( a ) 300 m ; ( b ) top 100 m ; insets 30 m .
( c ) plastic sections of a control ( 1 ) and perfused ( 2 ) eye showing the drainage structures .
images at lower magnification show the angle in the context of the iris and cornea ( top ) .
higher magnification images ( bottom ) allow for visualization of the open sc and tm .
we next determined if perturbation of outflow at a molecular level can be detected using our perfusion device .
we first tested the effect of the rock inhibitor , y-27632 ( see supplementary methods for details ) , on c. y-27632 is well established to increase outflow in other species .
we observed a 74.4% increase in c in eyes treated with y-27632 ( n = 10 , t - test p = 0.003 ; fig . 6 ) .
thus , as expected , rock inhibition increased c. this experiment validated the use of our system to measure changes in c. rho - associated protein kinase inhibitor y27632 increases outflow in mouse eyes .
eyes treated with y27632 were perfused with 200 m drug in bss for 30 minutes at 15 mm hg , followed by a 15 minutes of perfusion at 5 mm hg before measuring outflow ( protocol a ) .
the lines of fit for the linear part of the plots are : control , y = 0.0047x + 0.0175 , r = 0.99946 ; y27632 , y = 0.0082x + 0.0003 , r = 0.9996 .
c was different between groups : control , c = 4.7  1.2 nl / min / mm hg ( whole eye c = 10.4  2.64 nl / min / mm hg ) ; y27632 , c = 8.2  3 nl / min / mm hg ( whole eye c= 18.04  6.6 nl / min / mm hg ) .
nos3 ( endothelial nitric oxide synthase , enos ) and cav1 are genes implicated in glaucoma .
we first disrupted nos3 function by inhibition using the inhibitor l - name ; see supplementary methods for details ) as well as through genetic mutation .
treatment with l - name caused a reduction in c by 28.3% from control ( n = 7 eyes ; t - test , p = 0.025 ; fig .
c was reduced by 35.8% compared to control ( n = 7 eyes ; t - test , p = 0.008 ; fig .
next , we performed outflow measurements on cav1 null ( cav1 ) eyes ( n = 9 eyes ; fig . 8) . in cav1 eyes ,
c was reduced by 47.8% compared to control ( t - test , p = 0.02 ) .
we detected this reduction in two independent experiments on a b6 strain background and once on a mixed strain background ( not shown ) .
the requirement of nos3 activity was tested using nos3 mutant or l - name  treated wild - type eyes .
( a ) flow - rate versus pressure graph obtained from l - name  treated and control eyes .
( b ) flow - rate versus pressure graph obtained from nos3 and control eyes .
black diamonds represent control eyes and black triangles either l - name  treated eyes or nos3 eyes .
the equations representing the trendline for the linear part of the curve ( in [ a ] and [ b ] ) were : control , y = 0.0053x + 0.0254 , r = 0.9997 ; l - name , y = 0.0038x + 0.0382 , r = 0.99971 ; and nos3 
 y = 0.0034x + 0.0211 , r = 0.9976 .
c was different between groups : control , c = 5.3  1.4 nl / min / mm hg ( whole eye c = 11.6  3.08 nl / min / mm hg ) ; l - name , c = 3.8  0.7 nl / min / mm hg ( whole eye c = 8.36  1.54 nl / min / mm hg ) ; nos3 , c = 3.4  0.6 nl / min / mm hg ( whole eye c = 7.48  1.32 nl / min / mm hg ) .
flow - rate versus pressure graph showing a comparison of flow rates in control and cav1 eyes .
the equations representing the trend line for the linear part of the curve were : control , y = 0.007x + 0.0326 , r = 0.99322 , black circles ; cav1 , y = 0.0036x + 0.039 , r = 0.99322 , black diamond .
the computed c values were : control , c = 6.9  3.4 nl / min / mm hg ( whole eye c = 15.18  4.4 nl / min / mm hg ) , and c = 3.6  2.5 nl / min / mm hg ( whole eye c = 7.92  5.5 nl / min / mm hg ) .
we described a new way to study the molecular mechanisms controlling outflow . using a newly engineered in vitro perfusion system
, we first established a mechanistic role for the kinase activity of rock in mouse conventional outflow .
we also showed that nos3 and cav1 function , both previously implicated in glaucoma , is necessary for normal outflow .
these studies demonstrated the use of our system for identifying molecules and/or pathways controlling outflow .
several other investigators have described the nonlinearity of the flow rate  pressure plot above 25 mm hg .
recently , it has been determined that anterior chamber deepening with increasing pressure ( over 25 mm hg ) in enucleated mouse eyes results in traction on the tm , thus increasing c. our mounted eyes do not have an intact iris and ac , but similar nonlinearity was observed .
although the mechanism explaining this is not clear , increasing traction on the mounted tissue may itself allow nonlinear increases in outflow with increasing pressure .
a recent publication , using closely spaced pressure points and sophisticated data analysis , supports nonlinear changes in outflow with changing pressure .
a recent report suggested that insufficient hydration results in a positive y - intercept and that hydrating enucleated eyes by immersion eliminated this positive y - intercept , actually rendering it negative . during our experiments , we had maintained the mounted eyes in a humid environment ( measured to be 100% humidity ) to prevent evaporation .
nevertheless , our positive intercepts suggested that the humidity measurements might not have been accurate , allowing some evaporation .
thus , in response to this recent report , we performed our perfusion protocol after submerging eyes in pbs at 37c .
the submersion changed the y - intercept to 18.5 l / min , indicating that evaporation from the surface of the eye may be occurring in our humid environment .
importantly , the c value obtained when submerging the eyes was c = 5.065  1.1 nl / min / mm hg .
this value is not significantly different from that obtained when eyes were maintained in a humid atmosphere ( 4.7  1.2 nl / min / mm hg ; p = 0.49 ) .
this suggested that our original approach of maintaining the eye in the humid atmosphere did not affect measurement of c. importantly and during peer review of the current study , another study found no flow at 0 mm hg and suggested that negative intercepts are an artifact resulting from linear fitting of data .
this study sampled flow at nine pressures over the 5 to 20 mm hg range .
this dense sampling allowed power - fitting with a y - intercept of zero at 0 mm hg .
such dense sampling can be tested with our perfusion system and could improve accuracy in future experiments . to our knowledge , this is the first report that inhibiting rock activity increases conventional outflow in mouse eyes , as it does in other species , including human eyes .
our data suggested that the previous report of y27632 reducing iop in mice results from increased outflow .
rho - associated protein kinase has been implicated in a mechanosensing role in sc cells by controlling cell stiffness .
this is supported by additional studies where overexpression of human nos3 in mouse eyes resulted in increased outflow and l - name reduced outflow . on a mechanistic level ,
biomechanical strain on sc cells triggers activation / expression of nos3 function to elevate nitric oxide ( no ) levels .
given the ubiquitous expression of cav1 , it could be important for iop , function of retinal ganglion cells , and cells in the optic nerve head .
our data indicated that cav1 is at least required for conventional drainage and will impact iop .
given that we used a mutant mouse where cav1 is globally deleted , we will in the future perform cell type  specific inhibition to identify the critical cell type that controls outflow through cav1 function . the protein cav1 has been implicated in mechanotransduction and regulation of nos3 activity .
loss of either function ( not mutually exclusive ) would prevent translation of pressure - dependent strain on sc / tm cells into signals required for outflow . in summary
, we have successfully developed a new perfusion system to study molecular mechanisms controlling outflow .
we described a new role for cav1 in outflow physiology and provide more evidence for central roles for nos3 and rock in outflow regulation . with its potential for organ culture ,
 purposethe molecular mechanisms controlling aqueous humor ( aqh ) outflow and iop need much further definition . the mouse is a powerful system for characterizing the mechanistic basis of aqh outflow . to enhance outflow studies in mice 
 , we developed a perfusion system that is based on human anterior chamber perfusion culture systems . 
 our mouse system permits previously impractical experiments.methodswe engineered a computer - controlled , pump - based perfusion system with a platform for mounting whole dissected mouse eyes ( minus lens and iris , 45% of drainage tissue is perfused ) . 
 we tested the system 's ability to monitor outflow and tested the effects of the outflow - elevating drug , y27632 , a rho - associated protein kinase ( rock ) inhibitor . 
 finally , we tested the system 's ability to detect genetically determined decreases in outflow by determining 
 if deficiency of the candidate genes nos3 and cav1 alter outflow.resultsusing our system , the outflow facility ( c ) of c57bl/6j mouse eyes was found to range between 7.7 and 10.4 nl / minutes / mm hg ( corrected for whole eye ) . 
 our system readily detected a 74.4% y27632-induced increase in c. the nos3 inhibitor l - ng - nitroarginine methyl ester ( l - name ) and a nos3 null mutation reduced c by 28.3% and 35.8% , respectively . 
 similarly , in cav1 null eyes c was reduced by 47.8%.conclusionswe engineered a unique perfusion system that can accurately measure changes in c. we then used the system to show that nos3 and cav1 are key components of mechanism(s ) controlling outflow . 
Materials and Methods
Animals
Perfusion Apparatus
Mounting a Dissected Eye on Perfusion Apparatus
Monitoring and Control System
Outflow Measurements
Results
The Perfusion System
Characterization of the System: System Compliance and Resistance
Washout in Mouse Eye
Outflow Measurements
Inhibition of Rho-Associated Protein Kinase (ROCK) Increases Outflow
None
Discussion
Supplementary Material