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Elevated intraocular pressure (IOP) is primarily due to increased aqueous outflow resistance, but how aqueous outflow resistance is generated and regulated are still not fully understood. The aim of this study is to determine whether changes in outflow facility, outflow pattern, and morphology following acute IOP elevation were reversible when the IOP was returned to a normal level in bovine eyes using a two-color tracer technique to label outflow patterns within the same eye.
Twelve fresh enucleated bovine eyes were perfused with Dulbecco’s phosphate buffer saline (PBS) containing 5.5 mmol/L glucose (DBG) at 30 mmHg first to establish the baseline outflow facility followed by a fixed volume of red fluorescent microspheres (0.5 μm, 0.002% v/v). After the red tracer being replaced with DBG in the anterior chamber, perfusion was continued at 7 mmHg with the same volume of green tracer, followed by a fixative. In two control groups, the eyes were constantly perfused at either 30 mmHg (n=6) or 7 mmHg (n=6) using the same methods. The outflow facility (C, μl·min·−1mmHg−1), was continuously recorded. Confocal images were taken along the inner wall (IW) of the aqueous plexus (AP) in frontal sections. The percent of the effective filtration length (PEFL, PEFL=IW length exhibiting tracer labeling/total length of IW) was measured. Sections with AP were processed and examined by light microscopy. The total length of IW and the length exhibiting separation (SL) in the juxtacanalicular connective tissue (JCT) were measured. A minimum of eight collector channel (CC) ostia per eye were analyzed for herniations.
In the experimental (30–7 mmHg) group, the outflow facility was significantly higher at 7 mmHg ((4.81±1.33) μl·min·−1mmHg−1) than that at 30 mmHg ((0.99±0.15) μl·min·−1mmHg−1, P=0.002), corresponding to a significant increase in the PEFL (P=0.0003). The percent of CC ostia exhibiting herniations in the experimental group ((67.40±8.90) μl·min·−1mmHg−1) decreased significantly compared to that in the control at 30 mmHg ((94.44±3.33) μl·min·−1mmHg−1, P=0.03), but higher than that in the control at 7 mmHg ((29.43±4.60) μl·min·−1mmHg−1, P=0.01). Washout-associated separation between the IW and JCT was found by light microscopy and percent separation length (PSL, PSL=SL/total length of IW) was decreased in the control at 30 mmHg compared to that in the experimental group and control at 7 mmHg.
The pressure-induced morphological and hydrodynamic changes were reversible. Changes (collapse of AP, separation between the JCT and IW, and herniation into CC ostia) influence the effective filtration area that regulates outflow facility.
Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of primary open-angle glaucoma (POAG).1 The pressure rise is primarily due to increased aqueous outflow resistance,2,3 but how aqueous outflow resistance is generated and regulated is still not fully understood. Our group and others have recently demonstrated that drainage of aqueous humor out of the trabecular meshwork (TM) is not uniform throughout the anterior chamber (AC) angle but segmental in mouse,4 porcine,5 bovine,6–8 monkey,9,10 and human eyes.5,11 At any given time, only a fraction of the outflow pathways are actively involved in aqueous humor drainage. We have termed this active area the effective filtration area (EFA). Previous studies by our group have demonstrated that an increase in outflow facility (the inverse of outflow resistance) induced by both application of the Rho kinase inhibitor, Y27632 and washout12 (a volume-dependent increase in outflow facility after prolonged ocular perfusion13,14) correlated with an increase in EFA in bovine,7 and monkey,9 which is associated with a separation between the inner wall (IW) of Schlemm’s canal (SC) and juxtacanalicular connective tissue (JCT).
In contrast, acute and chronic elevation of IOP in bovine6 and monkey10 eyes correlated with a decrease in EFA, which is associated with collapse of SC, and herniation into the collector channel (CC) ostia. Additionally, an inverse correlation was reported between EFA and IOP in a secreted protein acidic and rich in cysteine (SPARC)-knockout ocular hypotensive mouse model.4 These studies suggest that structural changes in the JCT and IW of SC influence local outflow hydrodynamics that regulate outflow facility.
In this study, we hypothesize that hydrodynamic and morphological changes associated with decreased outflow facility following acute experimental elevation of IOP in normal eyes can be reversed when the IOP is returned to a normal physiologic pressure. To test our hypothesis, we investigated the relationship between the outflow facility and patterns at two different IOPs (30 mmHg first, then reduced to 7 mmHg) within the same eye using a previously developed two-color tracer technique8 with confocal microscopy, and further compared the morphological characteristics in this group with that in the control eyes that were perfused at either 30 or 7 mmHg only to determine whether the changes in outflow facility, pattern, and morphology following acute intraocular IOP elevation were reversible when the IOP was returned to a normal level.
Twelve pairs of fresh enucleated bovine eyes were obtained from a local abattoir (Arena and Sons, Hopkinton, MA, USA) and delivered on ice within 6 hours postmortem. Eyes with discernible damage or accumulated blood in the limbus or AC were excluded. The perfusion fluid was Dulbecco’s phosphate-buffered saline (Invitrogen, Grand Island, NY, USA) containing 5.5 mmol/L D-glucose (collectively referred to as DBG) that was passed through a 0.2 μm cellulose acetate filter prior to use.
Carboxylate-coated fluorescent microspheres (0.5 μm; Invitrogen, Grand Island, NY, USA) with emission/excitation wavelengths (red 580/605 nm, green 505/515 nm) suspended in DBG (0.002% v/v) were used in this study to trace changes in the aqueous outflow pattern. The size and concentration of the fluorescent microspheres were based upon a previous study by Johnson et al,15 who reported that particle sizes ranging from 0.176 to 0.460 μm at a concentration of 4×10−6 was dilute enough to not impede aqueous outflow through the JCT and IW in bovine eyes.
Details of the mechanical setup of the perfusion system were described previously.16 Briefly, the perfusion system consisted of a perfusion chamber and a collection chamber; the perfusion chamber was linked to a pressure transducer connected electronically to a computer control system. Outflow facility (C=Q/IOP) was measured at 10 Hz, ensemble averaged over a 10-second window, and electronically recorded every 10 seconds by LabView version 7.0 (National Instrument, USA).
Extraocular tissue was removed from bovine eyes, which were submerged to the limbus in PBS at 34°C. A 21-gauge infusion needle was inserted intracamerally through the peripheral transparent cornea into each eye and connected to the perfusion chamber. This needle was carefully threaded through the pupil and the needle tip positioned within the posterior chamber to prevent deepening of the AC that would otherwise lead to an artificial increase in outflow facility.17 A second needle was inserted intracamerally into the AC of each eye and connected to the collection reservoir. During the perfusion the collection reservoir tube was clamped except during exchanges. During exchanges, IOP was maintained at either 30 or 7 mmHg by raising the perfusion reservoir 2 mmHg higher than either 30 or 7 mmHg above the corneoscleral limbus, and decreasing the collection reservoir 2 mmHg lower than either 30 or 7 mmHg so that the contents of the AC would flow to the collection reservoir. A volume of 7 ml fluid was exchanged which took about 15 minutes. This amount (7 ml) was chosen for exchange based on our previous experiment, in which the amount of microspheres almost completely disappeared in the perfusate from the AC.
One eye of each pair (n=12) was used in the experimental group, in which the eyes were first perfused at 30 mmHg and then reduced to 7 mmHg (30–7 mmHg). The other eye was used as a control. Because bovine eyes exhibit washout,16 two control groups, consisting of perfusion pressure maintained at either 30 or 7 mmHg (30–30 and 7–7 mmHg; n=6 for each group), were included to account for the washout effect. Eyes in the experimental group (30–7 mmHg) and in the 30–30 mmHg control group were first perfused at 30 mmHg with DBG, while eyes in the 7–7 mmHg control group were perfused at 7 mmHg for at least 30 minutes to establish a stable baseline facility. A fixed volume of red fluorescent microspheres in DBG was filled (7 ml) into the AC and an additional 2 ml was then perfused. The red tracer was removed by exchanging the contents of the AC with DBG as described above. Perfusion pressure was then reduced to 7 mmHg in the experimental group, while the two control groups were maintained at either 30 or 7mmHg. A fixed volume of DBG (1.5 ml) was then perfused into the AC followed by a similar AC exchange and perfusion with green fluorescent microspheres in DBG. All eyes were perfusion-fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in PBS (pH 7.3) at the final perfusion pressure. During the entire perfusion procedure, the outflow facility was continuously recorded. After fixation, a 1 cm cut was made in the equator of each eye to ensure better fixation, and the eyes were then immersed in the same fixative and kept at 4°C until further processing.
All fixed eyes were cut in half at the equator, and the vitreous body and lens were carefully removed. The anterior segments were then divided into four quadrants (temporal, nasal, superior, and inferior). The 1.0–1.5 mm thick frontal sections of TM (tangential to the corneoscleral limbus and perpendicular to the ocular surface),6,7,18 were cut and counter-stained with To-pro3 (Invitrogen) for 30 minutes to visualize cell nuclei, followed by three successive five-minute washes in PBS. The sections were examined under a confocal microscope (Carl Zeiss 510, Axiovert 100M Laser Scanning Microscope, Heidelberg, Germany). A multi-track channel system was used to visualize the red and green fluorescent microspheres using 20× objective. At least six images per quadrant and 24 images per eye were taken randomly along the IW of the aqueous plexus (AP). The percent of the effective filtration length (PEFL; length of IW exhibiting tracer labeling/total length of IW) was measured as described previously.8 All measurements were performed by a trained investigator. These measurements were repeated again one month later. Intra-observer reliability for all measurements was within ±10%.
After confocal images being taken, sections that contained AP were further processed for conventional light microscopy. The sections were post-fixed in 2% osmium tetroxide in 1.5% potassium ferrocyanide for 2 hours, dehydrated in a graded series of ethanol, and embedded in Epon-Araldite. Semi-thin sections (3 μm) were cut and stained with 1% Toluidine Blue (Fisher Scientific Co., Pittsburgh, PA, USA). Light micrographs were taken along the IW of the AP by an Olympus BS40 light microscope with a Q-color 3 digit camera using 10× and 20× objectives to analyze the morphological changes in the different IOP groups. At least eight ostia in each eye were examined and graded as open, partially obstructed, or completely obstructed (Figure 1), by the IW and JCT protruding into the CC ostia (these are referred to as herniations) as described previously.6 The mean percentage of ostia with herniations was calculated in each group.
Washout induced outflow facility increase was previously reported to be associated with the separation between the IW and underlying JCT.7,12 Separation between IW endothelial cells and the underlying JCT was measured in all the light micrographs (Universal Desktop Ruler software, v2.9.1124, Figure 2). Percent separation length (PSL) was calculated as the ratio of the IW showing separation (SL) to the total length of the IW (TL) of the AP (PSL=SL/TL).
Data were shown as mean ± standard deviation (SD). A two-tailed unpaired Student’S t-test was applied using SPSS 11.0 (SPSS Inc., USA). P <0.05 was considered statistically significant.
Outflow facilities of the three groups were shown in Figure 3. The average baseline facility in the experimental (30–7 mmHg) group ((0.99±0.15) μl·min·−1mmHg−1) was not significantly different from that of the 30–30 mmHg control group ((0.90±0.19) μl·min·−1mmHg−1, P=0.373), while baseline outflow facility was significantly higher in the 7–7 mmHg control group ((2.16±0.44) μl·min·−1mmHg−1, P=0.001).
When the IOP in the experimental group was decreased from 30 to 7 mmHg, the mean outflow facility at 7 mmHg increased significantly compared to that at 30 mmHg ((4.81±1.33) vs. (0.99±0.15) μl·min·−1mmHg−1, P=0.002), which approached that in the 7–7 mmHg control group ((5.08±0.84) μl·min·−1mmHg−1, P=0.673), but was significantly higher than that in the 30–30 mmHg control group ((1.90±0.67) μl·min·−1mmHg−1, P=0.002).
In the two control groups, mean outflow facility were 117.25% (30–30 mmHg group) and 141.01% (7–7 mmHg group), respectively; higher at the dropdown point of the experimental group due to washout effect compared to that at baseline. The percentage of the increased outflow facility ((Cdropdown − Cbaseline)/Cbaseline × 100%) in the 30–7 mmHg group ((364.60±6.71)%) was significantly higher than either 30–30 mmHg control ((117.25±9.16)%, P <0.01) or 7–7 mmHg control ((141.01±7.10)%, P <0.01). No significant difference in the percentage of increased outflow facility was found between two control groups (P=0.597).
The outflow patterns labeled by two different colored fluorescent microspheres in the same eye at the same or different IOP are shown in Figure 4. In all the three groups, a segmental flow pattern was observed with a greater concentration of tracer in the TM near the CC ostia, and a larger area was labeled by green tracer than that labeled by red tracer, due to washout effect.12
In the 30–7 mmHg group (Figure 4A), the outflow pattern labeled by red tracer at 30 mmHg was often seen near the CC ostia region, while the outflow pattern labeled by green tracer became more diffuse along the AP after the pressure was dropped down to 7 mmHg. In the 30–30 mmHg control (Figure 4B), the outflow pattern was more segmental with both colors of fluorescent tracer appearing largely co-localized near the CC ostia region. In the 7–7 mmHg control (Figure 4C), a more uniform pattern was seen exhibiting diffuse co-localization along the IW of the AP compared to that in the 30–30 mmHg control.
The measurements of the effective filtration lengths are shown in Figure 5. In the 30–7 mmHg group, the PEFL labeled by the red fluorescent microspheres (PEFLred) at 30 mmHg ((12.86±5.89)%) was not significantly different from that of the 30–30 mmHg control ((11.49±4.95)%, P=0.734) but significantly lower than that of the 7–7 mmHg control ((28.05± 4.40)%, P=0.005).
The PEFL labeled by green fluorescent microspheres (PEFLgreen) was significantly larger than the PEFLred in all three groups due to washout effect.12 However, the difference between PEFLgreen and PEFLred in the 30–7 mmHg was significantly larger ((45.03±4.01)%) than that of either the 30–30 mmHg control group ((25.85±5.60)%, P=0.003) or the 7–7 mmHg control group ((25.47±7.81)%, P=0.003), but no significant difference was found between the two control groups (P=0.966).
In the 30–7 mmHg group, the PEFLgreen increased to (57.89±8.45)%, which was not significantly different from that of the 7–7 mmHg control group ((53.96±9.94)%, P=0.527) and was significantly higher than that of the 30–30 mmHg control group ((37.34±8.17)%, P=0.013).
Light microscope images near the CC ostia region in the three IOP groups are shown in Figure 6. In the 7–7 mmHg group (Figure 6A), the AP was open in most areas. Giant vacuoles18 along the AP and separation between the IW and underlying JCT were observed; the TM and the JCT were loose. In the 30–7 mmHg group (Figure 6B), the lumen of the AP was partly collapsed. Both separation between the IW and JCT and giant vacuoles could only be observed in the area where the AP was open. The IW and JCT were partially herniated into the CC ostia. At the higher IOP, 30–30 mmHg group (Figure 6C), the AP was completely collapsed in some areas. Less separation between the IW and JCT and denser TM and JCT were seen in the area where the AP collapsed. Complete herniations into CC ostia were commonly observed in this group.
The average percent of CC ostia demonstrating herniations in each of the three groups are summarized in Table 1. In the 30–7 mmHg group, the average percent of CC ostia exhibiting herniations ((67.40±8.90)%) decreased significantly compared to that of the 30–30 mmHg control group ((97.48±3.01)%, P=0.025), while it was still higher than that of the 7–7 mmHg control group ((30.32±4.75)%, P=0.016). The percentage of complete herniations ((4.20±3.19)%) in the 30–7 mmHg group significantly decreased compared to that in the 30–30 mmHg control group ((51.69±3.88)%, P <0.01), but was similar to that in the 7–7 mmHg control group ((2.08±2.26)%, P=0.668).
The percentages of the separation length (PSL) in the three groups are shown in Figure 7. The PSL of 30–7 mmHg group was 38.32±3.57, which is significantly larger than that of the 30–30 mmHg group (17.24±1.32, P=0.009), but significantly smaller than that of the 7–7 mmHg group (51.55±5.66, P=0.041).
In this study, we analyzed the hydrodynamic changes of the aqueous outflow pathway following acute experimental variation of IOP within the same eye using a two-colored fluorescent tracer perfusion technique and confocal microscopy. After confocal images were taken, the same tissue was processed with light microscopy to correlate the structural changes and changes in the outflow pattern to better understand the mechanism of IOP related changes in outflow facility. Our results revealed that the decrease in outflow facility associated with acute elevation of IOP was reversible when elevated IOP was reduced to a normal level.
The changes in the outflow facility with IOP variation were associated with that in the available area for aqueous outflow, which in turn were regulated by morphological changes. Using our previously developed two-colored tracer perfusion method,8 the following reversible morphological changes were observed. With increasing IOP, the pressure compressed the TM resulting in collapse of the AP. In the area of AP collapse, little or no tracer was observed suggesting little or no flow across the IW of the AP in these regions. This could be due to lack of sufficient space for the IW endothelial cells to form giant vacuoles18 and/or expand or separate from the underlying JCT, resulting in an increase of outflow resistance in these areas. This morphological change resulted in that the tracer flowed to a non-collapsed area.
In the CC ostia regions, a greater pressure difference existed19 and since there was no outer wall limitation, the IW and underlying JCT were able to expand or herniate into the CC ostia regions. Although an increase in the numbers and degree of herniations into CC ostia was found to be associated with increasing IOP, the outflow, identified by tracer labeling, was still able to pass through these herniated regions. However, because the collapse of the AP confined the outflow to the CC ostia regions, the total activated filtration area was decreased, which may contribute to a decrease in outflow facility. When the IOP was decreased from a high to normal level, the collapsed AP reopened, thus allowing room for more giant vacuoles to form as well as separation between the IW and JCT. The separation between the IW and the JCT may eliminate the funneling effect20 (which refers to the connection between the IW and JCT refining the aqueous outflow to special areas with pores in the IW) and cause decreased outflow resistance in these areas. Additionally, the blockage of CC ostia by the herniated IW and JCT tissue was partially or completely reversed. Such changes contributed to the increase in the available area for outflow and explained the reversion of outflow facility when the IOP was decreased from a high to normal level. In our experimental group, when the IOP was decreased from 30 to 7 mmHg, the total number of ostia exhibiting herniations was higher, and the percentage of the separation length was less than those in the 7–7 mmHg control. This may be due to a shorter perfusion time after the IOP was decreased. However, this incomplete reversal did not keep the outflow facility from returning to a level similar to that of the 7–7 mmHg control. This could be due to the reversal of AP collapse near the CC ostia region, and thus EFA was increased when the herniations changed from being almost complete (in 30 mmHg) to partially complete (in 7 mmHg). Furthermore, when the IOP was decreased, the TM became less compact and the AP reopened in more areas resulting in the EFA returning to the same level as that of the 7–7 mmHg control group. These data support our hypothesis that hydrodynamic and morphological changes associated with decreased outflow facility following acute experimental elevation of IOP in normal eyes are reversible when the IOP is returned to a normal physiologic pressure. Our findings also suggest that pressure-induced morphological changes need time to be reversed. If the IOP remained at a higher level chronically, these morphological changes may gradually become irreversible, which has been reported in POAG eyes.21 In the future, an animal model with ocular hypertension need to be developed and the morphological changes at different time point of the ocular hypertension are promised to be addressed.
Fresh enucleated bovine eyes were used in this study because of their availability, which offered a practical way to achieve the aims of this study. It has been well established that bovine eyes exhibit washout during long-term perfusion.14,16,22,23 To account for the washout effect, two control groups were included in which two colored tracers were perfused at either 30 or 7 mmHg continuously to estimate the washout-induced changes in the outflow patterns before and after washout at the same pressure. Although both the outflow facility and the PEFL increased between the first and second tracer in the two control groups due to the washout effect, the percentage of increase in the outflow facility and the PEFL in our experimental (30–7 mmHg) group was significantly higher than that in both control groups. A decrease in IOP would likely account for these additional changes in the outflow facility and the PEFL.
In summary, using the two-colored tracer perfusion method, we were able to discover the reversal of hydrodynamic and morphological changes related to changes in outflow facility within the same eye. The pressure-induced structural changes, such as progressive collapse of the AP and an increasing number of the herniation into CC ostia caused a decrease in the available area for outflow, which contributed to decreased outflow facility. These morphological changes and their resulting outflow facility decrease were partially or completely reversed when the IOP was decreased from a high to normal level in acute experimental conditions. Our previous study21 found that in enucleated POAG eyes, similar morphological changes become irreversible even when the eyes were fixed at 0 mmHg. Further studies are needed to understand the mechanism of how the reversible IOP-related structural changes of normal human eyes in acute experimental conditions become irreversible in the POAG eyes.
This study was supported by grants from American Health Assistance Foundation (NIH-EY018712), Wing Tat Lee Fund from Boston University School of Medicine, Massachusetts Lions Eye Research Fund, Young Scholars Supporting Fund from School of Medicine, Fudan University and Huadong Hospital Fundation.
The authors thank Stephanie Battista, OD for the preliminary work in development of the perfusion technique and Rozanne Richman for technical assistance.
ZHU Jing-ying, Department of Ophthalmology, Huashan Hospital of Fudan University, Shanghai 200040, China. Department of Ophthalmology, Huadong Hospital of Fudan University, Shanghai 200040, China. Department of Ophthalmology, Boston University School of Medicine, Boston, MA 02118, USA.
YE Wen, Department of Ophthalmology, Huashan Hospital of Fudan University, Shanghai 200040, China.
WANG Ti, Department of Ophthalmology, 85th Hospital of People’s Liberation Army, Shanghai 200052, China.
GONG Hai-yan, Department of Ophthalmology, Boston University School of Medicine, Boston, MA 02118, USA.