Morphological And Hydrodynamic Correlates For Changing Aqueous Humor Outflow Resistance

PartⅠHydrodynamic and Morphological Correlates in Monkey Eyeswith Laser Induced-GlaucomaPurpose:To investigate the relationship between decreased outflow facility and thechanges in hydrodynamic aqueous outflow pattern and morphology after chronicelevated intraocular pressure (IOP)in laser-induced glaucoma of cynomolgusmonkey eyes.Methods:Argon laser photocoagulation bums to the trabecular meshwork (TM)were made in one eye of each monkey (N=3),leaving the contralateral eye as anormotensive control.Between 16 and 70 months later (mean±STD=44.32±27.10),measurements were made of IOP by pneumatonometry and outflow facility byfluorophotometry.To label the hydrodymamic patterns of outflow,the eyes wereenucleated and perfused with fluorescent microspheres (0.5μm;0.002%)at the lastpressure measured before death minus 7mmHg.The eyes were perfusion-fixed withKarnovsky's fixative at the same pressure.Confocal images were taken along theinner wall (IW)of the Schlemm's canal (SC).The total length (TL)and the filtrationlength (FL)of the IW decorated by tracers were measured in frontal sections.Theaverage percent effective filtration length (PEFL=FL/TL)was calculated for eacheye.Sections exhibiting SC were then processed and examined under light andelectron microscopy.Results:The average IOP in laser-treated eyes was 61.33±4.16 mmHg,which wassignificantly higher than that of controls (22.67±4.04 mmHg,P=0.0003).Theaverage outflow facility (0.03±0.02μl/min/mmHg)was 13-fold lower inlaser-treated eyes than that of controls (0.39±0.17μl/min/mmHg,P=0.02).Byconfocal microscopy,in control eyes,SC was open and a segmental distribution ofmicrospheres was found with a greater concentration in the juxtacanalicularconnective tissue (JCT)region near the collector channel (CC)ostia.Much lesstracer labeling along SC in laser-treated eyes was seen than that in controls.Theaverage PEFL in controls (47.47±10.79%)was 6-fold larger than in laser-treatedeyes (8.40±4.81%,P=0.013).By light microscopy,there was extensivepigmentation throughout the TM,denser extracellular matrix in the JCT region,andmost of the SC collapsed with focal herniations of the IW and JCT protruding into the CC ostia in laser-treated eyes.The mean width of SC in control eyes(19.14±5.75μm)was 4-fold wider than in laser-treated eyes (4.57±3.25μm,p＜0.0001).In the laser-treated eyes,16 CC ostia were examined,herniations werefound in 14 CC ostia (88%),while herniations were found in 4 CC ostia of 23 CCostia examined in control eyes (17%,p＜0.0001).By electron microscopy,few or nomicrospheres were observed in laser-treated areas.However,in non-lasered areas,numerous microspheres were observed near the CC ostia area compared withcontrols.Conclusions:In laser-induced glaucoma model,laser damage results in areduction in the available area for outflow across the IW of SC which contributes tothe decrease in outflow facility and thus elevation of the IOP Collapse of SC,causedby chronic elevation of IOP,further decreases in the available area for outflow in avicious cycle.This study suggests that available area for aqueous outflow play animportant role in regulating outflow resistance. PartⅡMorphological and Hydrodynamic Correlates for ChangingAqueous Humor Outflow Resistance with different IOPExperimentⅠPurpose:To understand how hydrodynamic and morphologic changes in theaqueous humor outflow pathway contribute to decreased aqueous humor outflowfacility following acute elevation of intraocular pressure (IOP)in monkey eyes.Methods:Enucleated monkey eyes were perfused at two different pressures30mmHg and 7 mmHg,while outflow facility was continuously recorded.Dulbecco's PBS + 5.5 mM glucose containing fluorescent microspheres (0.5μm,0.002% v/v)was perfused to outline aqueous outflow patterns followed byperfusion-flxation.Confocal images were taken along the inner wall (IW)of the SCin radial and frontal sections.Percent effective filtration length (PEFL=IW lengthexhibiting tracer labeling/total length of IW)was measured.Herniations of IW intocollector channel (CC)ostia were examined and graded for each eye by lightmicroscopy.Results:Increasing IOP from 7 to 30 mmHg coincided with a 2.3-fold decrease inoutflow facility,from 0.34±0.08μl/min/mmHg at 7 mmHg to0.16±0.06μl/min/mmHg at 30 mmHg (P=0.03),a 1.6-fold decrease in PEFL withtracer more confined to the vicinity of CC ostia,from 56.02±6.04% at 7 mmHg to36.08±0.38% at 30 mmHg (P=0.03).Progressive collapse of the SC and increasingpercentage of CC ostia exhibiting herniations at 30 mmHg under light microscopy.Conclusions:Decreasing outflow facility during acute IOP elevation coincides witha reduction in available area for aqueous humor outflow and confinement of outflowto the vicinity of CC ostia.These hydrodynamic changes are likely driven bymorphologic changes associated with SC collapse and herniation of IW of SC intoCC ostia. ExperimentⅡPurpose:To determine whether the filtration area for aqueous outflow increaseswhen IOP is decreased from 30mmHg to 7mmHg or from 45mmHg to 7mmHgwithin the same eye.Methods:We used two tracers with different excitation and emission spectra inorder to label the outflow patterns before and after reducing the perfusion pressure.Ten freshly enucleated monkey eyes were perfused at 30 mmHg (n=4)with 0.3 mlof red fluorescent microspheres (0.5μm,0.002% v/v),followed by an anteriorchamber exchange to remove the red tracer.A perfusion was continued at 7 mmHgwith a green tracer (0.3ml),followed by a pefusion with Karnovsky's fixative(0.3ml).The control eyes were perfused with only red tracer (0.3ml)at either 30mmHg (n=3)or 7 mmHg (n=3).Sections were cut radially and tangentially to thelimbus in all four quadrants and counter-stained for laser confocal.The pattern offluorescent tracer distribution was visualized in the trabecular meshwork,SC andCC ostia.Outflow facility and effective filtration area were measured at 30mmHgand 7mmHg in separate eyes (control)as well as within the same eye in which thepressure was decreased (experimental).Sections displaying the SC and CC ostiawere then processed for light micrscopy to analyze and grade the numbers of CCostia obstructed by herniated IW and JCT.Two human eyes were perfused with twodifferent tracer in the same way,one was kept the IOP at 45mmHg as control,theother is from 45mmHg to 7mmHg,then observed under confocal and lightmicroscopy.Results:As the perfusion pressure decreased from high to normal within the sameeye the herniations were found to be either partially or completely reversible,andcorrelate with an increase in facility as well as an increase in the area available foroutflow either in monkey eyes or in human eyes.The average facility ofexperimental eyes perfused at 30mmHg was 0.37±0.10μl/min/mmHg which waslower than the average facility measured after the perfusion pressure was dropped to7 mmHg (0.48±0.21μl/min/mmHg,P=0.6).The percent of ostia obstructed byherniated tissue in eyes that had an acute IOP decrease (from 30 mmHg to 7 mmHg,33.06±2.66%)and was lower than eyes perfused at a constant 30 mrnHg(79.04±20.50%,P=0.06)but not as low as in eyes that were perfused at a constant 7mmHg (P=0.6).Conclusions:We found that the herniations occurring in eyes that underwent anacute IOP increase are partially reversible and correlate with an increase in effectivefiltration area in the outflow pathway,as well as outflow facility.These studies pointto the importance of effective filtration length and the presence or absence ofhemiations as important factors in outflow resistance. PartⅢHydrodynamic and Morphological Changes during Washout inMonkey EyesPurpose:To better understand the physiology of the washout effect in monkey eyesin order to exploit it as a means of reducing IOP in human eyes with glaucoma.Methods:Eight enucleated monkey eyes were used in this study.Within 24 hourspostmortem,eyes were perfused with Dulbecco's PBS containing 5.5mM glucose at15mmHg for long-duration (180 minutes)or short-duration (30 minutes,n=4 foreach group).The eyes were perfused with red fluorescent microspheres (0.5μm;0.002% v/v)to trace the hydrodynamic patterns of outflow,and then wereperfusion-fixed with Karnovsky's fixative at the same pressure.Radial and frontalsections in all quadrants were prepared and confocal irnages were taken along theinner wall (IW)of the Schlemm's canal (SC).The total length (TL)and thetracer-decorated length (L)of IW were measured in＞16 images/eye,and theaverage percent effective filtration length (PEFL=L/TL)was calculated for each eye.The sections with SC were processed and examined under light and electronmicroscopy.The TL of IW and the length exhibiting separation (SL)injuxtacanalicular connective tissue (JCT)were measured.The average percentseparation length (PLS=SL/TL)was calculated.Results:Outflow facility increased 150% [1.46±0.70μl/min/mmHg (mean±SE),p＜0.01] from baseline facility (0.58±0.20μl/min/mmHg)after 180 minutesperfusion to induce washout.No significant increases were found in outflow facilityafter 30 minutes perfusion (0.44±0.16μl/min/mmHg)compared to baseline(0.62±0.21μl/min/mmHg,P=0.46).A more uniform tracer pattern was seen inlong-duration perfusion eyes than in short-duration perfusion eyes.The averagePEFL in long-duration eyes (83.37±4.13%)was 3.4-fold larger than inshort-duration eyes (24.19±8.42%,P＜0.001).The JCT region appeared distended inlong-duration eyes compared with short-duration eyes.The connections betweenJCT cells and between JCT cell and matrix were lost in expansion regions.The PLSin JCT region was 2.3-fold larger in long-duration eyes (77.4±3.2%)than that inshort-duration eyes (33.5±5.3%,p＜0.001).A significant positive correlation wasfound between PEFL and PSL (P=0.00002)suggesting that as connectivity betweenthe JCT and IW decreases the available area for aqueous humor drainage increasesalong the SC.Conclusions:Increasing outflow facility during washout coincides with an increasein available area for aqueous humor outflow.These hydrodynamic changes arelikely driven by morphologic changes associated with a decrease in cell-cell and cell-matrix connections in the JCT region.