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Elevation of intraocular pressure is usually associated with primary open angle glaucoma and caused by increased outflow resistance. A two-color fluorescent tracer technique was developed to investigate the hydrodynamics of aqueous humor outflow with changing intraocular pressure within the same eye, to better understand the relationship between outflow facility and effective filtration area.
Eighteen enucleated bovine eyes were first perfused at 30 mmHg with Dulbecco’s phosphate-buffered saline containing 5.5 mmol/L D-glucose. After a stable baseline facility, red fluorescent microspheres (0.5 μm, 0.002% v/v) were exchanged and perfused. Eyes in the one-color control group (n=6) were immediately perfused with fixative. In the experimental group (n=6), eyes were perfused with green tracer after intraocular pressure reduced to 7 mmHg, while in the two-color control group (n=6), eyes were perfused with green tracer with intraocular pressure remaining at 30 mmHg. All 12 eyes were then perfusion-fixed. Outflow facility was continuously recorded in all eyes. Confocal images were taken along the inner wall of the aqueous plexus and the percent of the effective filtration length (PEFL; length of inner wall exhibiting tracer labeling/total length of inner wall) was measured. The relationships between outflow facility and PEFL were analyzed statistically.
No significant differences were found in baseline facilities (μl·min−1·mmHg−1) among the three groups (the experimental group: 0.93±0.12; the two-color control group: 0.90±0.19; the one-color control group: 0.98±0.13). In the experimental group, the outflow facility was significantly higher at 7 mmHg (4.29±1.01) than that at 30 mmHg (1.90±0.67, P <0.001), which corresponded to a significant increase in the PEFL at 7 mmHg (54.70±8.42) from that at 30 mmHg ((11.76±4.56)%, P <0.001). The PEFL labeled by red fluorescent microspheres in the experimental group ((11.76±4.56)%) showed no significant difference from that of the one-color control group ((13.39±2.19)%, P=0.473) or the two-color control group ((11.49±4.95)%, P=0.930). The PEFL labeled by green fluorescent microspheres in the experimental group ((54.70±8.42)%) was significantly higher than that of the two color control group ((37.34±8.17)%, P=0.010). A positive correlation was found between outflow facility and PEFL(r=0.897, R2=0.804) in the experimental group.
Changes in aqueous humor outflow patterns before and after a change in intraocular pressure can be successfully distinguished within the same eye using our newly developed two-color tracer perfusion technique. The PEFL showed positive correlation with the outflow facility.
Primary open angle glaucoma (POAG), a leading cause of blindness, affects over 70 million people worldwide.1,2 Elevation of intraocular pressure (IOP), a primary risk factor in POAG, is mainly caused by increased aqueous outflow resistance.3–6 Several studies have localized the majority of outflow resistance to the inner wall (IW) endothelium of Schlemm’s canal (SC) and its underlying juxtacanalicular connective tissue (JCT),7–9 but the hydrodynamic details of how aqueous humor flows through these tissues and how these tissues generate outflow resistance in normal and POAG eyes are still not fully understood. Previous tracer studies at both confocal and electron microscope levels10–12 have concluded that aqueous humor outflow is “segmental” rather than uniform, so that only a fraction of the total area of JCT and the IW is active in aqueous humor filtration at any given time. This area is termed the effective filtration area, which was found to be proportionally related to the outflow facility11,12 and to decrease with experimentally acute and chronic IOP elevation.11–13 In a previous study using cationic ferritin, glaucomatous eyes appeared to exhibit a decreased effective filtration area compared with normal eyes at the electron microscope level.10 These findings suggest that the effective filtration area through the most resistive tissues of the IW and JCT may play a role in regulating the aqueous outflow resistance.
The goal of this study is to develop a two-color fluorescent tracer technique to investigate the relationship between the aqueous humor outflow facility and effective filtration area through the JCT and IW of the same eye before and after a change in IOP. Fresh enucleated bovine eyes were used because of their availability, offering a practical means to develop a new technique. The novelty of this technique is that by perfusing two different colored fluorescent microsphere tracers, each perfused at different periods of the experiment, we can determine if there is a distinguishable change in the effective filtration area before and after a change in IOP. This technique is more advantageous than the one-color tracer technique we used previously,11,12 because it offers its own internal control by showing tracer labeling pattern changes after the change in IOP within the same section of the same eye. This technique would greatly minimize the number of histological sections needed for analysis and avoid regional outflow variability between different eyes. Therefore, this would greatly simplify statistical analysis. If proven effective, this technique could be applied for further study in the human eye.
Eighteen enucleated bovine eyes (unpaired) were obtained from a local abattoir (Arena and Sons, Hopkinton, MA) and delivered on ice within 6 hours postmortem. Eyes with discernible damage or accumulated blood in the limbus or anterior chamber were excluded. All studies adhered to the association for research in vision and ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.
The perfusion fluid, Dulbecco’s phosphate-buffered saline (Invitrogen, Grand Island, NY, USA) containing 5.5 mM D-glucose (collectively referred to as DBG) 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 of 580/605 nm (red), or 505/515 nm (green), suspended in DBG (0.002% v/v) were used to trace changes in the aqueous outflow pattern. The size and concentration of fluorescent microspheres used in this study were based upon those of a previous study by Johnson et al,14 who reported that particle sizes ranging from 0.176 to 0.460 μm at a concentration of 4×10−6 were sufficiently dilute to not impede aqueous outflow through the JCT and IW in bovine eyes.
The perfusion apparatus used in this study consisted of a computer control system, a syringe pump, a pressure transducer, an exchange reservoir and a collection reservoir (Figure 1). Details of the mechanical setup of the perfusion system have been described previously.15 Briefly, the computer-controlled syringe pump delivered a variable flow rate (Q), to the anterior chamber to maintain a desired IOP that was monitored by a pressure transducer connected electronically to the computer control system (Lab View 7.0, National Instrument, USA). Outflow facility (C=Q/IOP) was measured at 10 Hz, ensemble averaged over a 10-second window, and electronically recorded every 10 seconds. The exchange chamber and a collection chamber were used to exchange the anterior chamber contents.
Bovine eyes were cleaned of extraocular tissue. Each eye was placed in a beaker, submerged to the limbus in phosphate-buffered saline (PBS), and the beaker was placed in a water bath (34°C). A 21-gauge infusion needle was inserted intracamerally through the peripheral transparent cornea into each eye, and was connected to the syringe pump and the exchange reservoir by a stop-cock and pressure tubes. The needle was carefully threaded through the pupil with the needle tip positioned within the posterior chamber, to prevent deepening of the anterior chamber that would otherwise lead to an artificial increase in outflow facility.16 A second needle was inserted intracamerally into the anterior chamber of each eye and connected to the collection reservoir. The collection reservoir tube was clamped throughout perfusion except during exchanges. During the exchange, IOP was maintained at either 30 or 7 mmHg by raising the exchange reservoir 2 mmHg higher (32 or 9 mmHg) above the corneoscleral limbus, and lowering the collection reservoir 2 mmHg lower (28 or 5 mmHg) so that the contents of the anterior chamber would flow to the collection reservoir.
Eighteen enucleated bovine eyes (unpaired) were randomly divided into three groups (experimental, one-color tracer control and two-color tracer control groups, n=6 each). These three groups were set up differently to account for variability. First, the experimental group (30–7 mmHg) had red fluorescent microspheres perfused at 30 mmHg, and green fluorescent microspheres perfused at 7 mmHg. Second, we included a two-color control group (30–30 mmHg) to account for the washout effect (outflow facility increased with prolonged perfusion time and increased volume of perfusate),17–19 with its perfusion pressure maintained at 30 mmHg for both tracers (red and green). Lastly, the one-color control group (30 mmHg) was included to exclude the possibility that the first red tracer might be washed away during perfusion of the second tracer.
All eyes were first perfused at 30 mmHg with DBG for a minimum of 30 minutes to establish a baseline facility, followed by exchanging a fixed volume (7 ml) of red fluorescent microspheres in DBG into the anterior chamber (AC) and perfusing for an additional 2 ml with the same tracer. For the experimental and two-color control groups, 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 that of the two-color control group was maintained at 30 mmHg. A fixed volume of DBG (1.5 ml) was then perfused into the AC and a second baseline facility was noted at this time. Green fluorescent microspheres in DBG were then exchanged and perfused using the same procedures as used for the red tracer. All 12 eyes were perfusion-fixed with 0.75 ml fixative (2.5% glutaraldehyde and 2% paraformaldehyde in PBS, pH=7.3) at the final perfusion pressure. For the one-color control group, after red tracer perfusion the eyes were immediately perfusion-fixed at 30 mmHg using the same protocol.
During the entire perfusion procedure, the outflow facility was continuously recorded in each eye. After fixation, a 1 cm cut was made in the equator of each eye to ensure better fixation, the eyes were then immersed in the same fixative overnight, transferred to PBS 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 4 quadrants (designated as temporal, nasal, superior and inferior). Frontal Sections (1–1.5 mm) of the trabecular meshwork (TM; tangential to the corneoscleral limbus and perpendicular to the ocular surface) were cut and counter-stained with To-pro3 (Invitrogen, Grand Island, NY, USA) for 30 minutes to visualize cell nuclei and this was 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 a 20× objective. At least 6 images per quadrant and 24 images per eye were taken randomly along the IW of the aqueous plexes (AP; equivalent to the Schlemm’s canal20).
The IW labeled by tracers was considered to be the effective filtration area.11,12 The length of the IW exhibiting either red (Lred) or green (Lgreen) tracer labeling and the total length (TL) of the IW of the AP, was measured using software LSM 510 version 3.2PS2 (Figure 2). The percentage of effective filtration length (PEFL) at different IOPs was then calculated as the ratio of the length of the tracer labeling (Lred or Lgreen) to the TL of the IW of the AP (PEFL = Lred/TL, Lgreen/TL).
A two-tailed Student’s t-test and a two-tailed Pearson’s test were applied with a required significance level of 0.05. Data were shown as means±standard deviation (SD).
Outflow facilities of the three groups were shown in Figure 3. The average baseline facility (μl·min−1·mmHg−1) in the experimental (30–7 mmHg) group (0.93±0.12) was not significantly different from the baseline facilities of either the two-color control group (0.90±0.19; P=0.688) or the one-color control group (0.98±0.13; P=0.521).
When the IOP in the experimental group was decreased from 30 to 7 mmHg, the second baseline outflow facility at 7 mmHg increased significantly compared with the baseline facility at 30 mmHg ((4.29±1.01) versus (0.93±0.12) μl·min−1·mmHg−1, respectively; P <0.001). Although the second baseline outflow facility in the two-color control group was significantly increased from its initial baseline facility ((0.90±0.19) versus (1.90±0.67) μl·min−1·mmHg−1; P=0.015) due to the washout effect, a significant difference was still observed in the second baseline facilities between the experimental group and the two-color control group ((4.29±1.01) versus (1.90±0.67) μl·min−1·mmHg−1 respectively; P=0.001).
The outflow patterns labeled by one or two-color fluorescent microspheres at different IOPs in frontal sections of the same eye were shown in Figure 4. In the two control groups (Figure 4A–C and 4G), the AP was narrow and collapsed; the IW of the AP and the underlying JCT were protruded or even herniated into the collector channel (CC) ostia. A segmental flow pattern labeled by fluorescent microspheres was observed with a greater concentration of tracer in the TM near the CC ostia.
In the experimental group (Figure 4B), the outflow pattern labeled by red tracer at 30 mmHg was concentrated near the CC ostia region while the outflow pattern labeled by green tracer at 7 mmHg became more diffuse along the AP. The AP in this group became wider than that of the control groups and less herniation of the CC ostia was observed.
The measurements of the EFL were shown in Figure 5. The PEFL labeled by red fluorescent microspheres (PEFLred) in the experimental group ((11.76±4.56)%) showed no significant difference from either the two-color control group ((11.49 ± 4.95)%, P=0.930) or the one-color control group ((13.39±2.19)%, P=0.473).
Due to the washout effect and/or IOP variation, the PEFL labeled by green fluorescent microspheres (PEFLgreen) was significantly greater than the PEFLred in both the experimental group and the two-color control group. However, even after accounting for the washout effect, the PEFLgreen was significantly higher in the experimental group ((54.70±8.42)%) than in the two-color control group ((37.34±8.17)%, P=0.010).
Using the two-tailed Pearson correlation test, a positive correlation was found between the outflow facility and PEFL in the experimental group (r=0.897, R2=0.804). The corresponding linear regression formula was Y=4.172+11.121X (X: PEFL, Y: outflow facility) (Figure 6).
In this study, we developed a two-color fluorescent tracer perfusion technique to investigate the relationship between the aqueous humor outflow facility and effective filtration area through the JCT and IW of the same eye, before and after a change in IOP. Our results demonstrated that the changes in the aqueous humor outflow patterns before and after a change in IOP can be successfully distinguished within the same eye using this newly-developed technique.
Advantages of using fluorescent tracers observed under confocal microscopy are: 1) it requires minimal tissue preparation and the same tissue can be further processed for electron microscopy after confocal images are taken; 2) a much larger area of the TM tissue can be visualized compared to previous studies using cationic ferritin as a tracer to label the aqueous outflow pathways which is only observable at the electron microscopic level.10,21,22 Furthermore, previous studies at the electron microscopic level only examined a small region of the TM tissue in radial (meridional) sections, leaving the location of the examined tissue unknown with regard to the collector channel (CC) ostia, the area where preferential flow was reported.11,23,24 Our previous11,13 and current studies demonstrated that aqueous outflow patterns are segmental and appear to be associated with CC ostia. Tracers used at the electron microscopic level make it difficult to appreciate the outflow patterns circumferentially over the entire TM.
By analyzing the confocal images and studying the relationship between the outflow facility and the effective filtration area, we found that the AP collapsed at the higher IOP levels and the CC ostia were herniated by the IW and underlying JCT. These morphological changes confined most of the red tracers near the CC ostia region, leading to a decreased effective filtration area, which was associated with decreased outflow facility. When the IOP was then decreased, the AP reopened, the herniations of the CC ostia were reversed and an increase in the effective filtration length was observed. These were found to be correlated with an increased outflow facility. Thus, our data demonstrated that the outflow facility increased with decreased IOP and was positively correlated with the effective filtration area. Compared with previous single tracer perfusion studies,11–13,16 the two-color fluorescent microsphere perfusion provided a feasible approach to observe the outflow pattern changes under different IOPs in the same eye and their relationship to the outflow facility. Therefore, using this newly developed technique, we can further explore the potential relationship between outflow facility and the outflow pattern under other different experimental conditions within the same eye. One such application could be studying the effect of different pharmacological agents.
The two colors of fluorescent microspheres chosen in our study could clearly label the outflow patterns with different IOP levels, which could be directly observed using confocal microscopy. This novel technique allowed us to explore the potential relationship between outflow facility and outflow pattern under different IOPs within the same eye. Compared with the previous studies using tracers, which could only be observed under the electron microscope, this newly-developed technique used in our study provided a larger scale of observation, simple processing procedures and more variable experimental conditions.
In the process of developing this technique, first, we needed to demonstrate that during perfusion of the second tracer, the first tracer would not be washed away. We therefore included a one-color control group. When comparing the PEFL of the red tracer with the experimental, the one-color control and the two-color control groups, we did not find significant differences in the PEFL of the red tracer among the three groups. These results suggest that the first color tracer was not significantly washed away during the perfusion of the second tracer.
Second, although the outflow pathway of bovine eyes has structures similar to human eyes, such as the AP, CC, JCT and TM, it has been well established that non-human mammalian eyes exhibit washout during long term perfusion.17,19,25 To account for the washout effect, a two-color tracer control group was included in which red and green tracers were sequentially perfused at the same pressure (30 mmHg) to estimate the washout-induced changes in the outflow patterns. Due to the washout effect, the second baseline outflow facility was found to be significantly higher than the initial baseline facility, and the PEFL of the green tracer was significantly larger than the PEFL of the red tracer in this control group. Even after accounting for the washout effect, the second baseline outflow facility and the PEFL of the green tracer were still significantly lower in the two-color control group than in the experimental (30–7 mmHg) group. Therefore, a decrease in IOP in the experimental group would likely account for the additional increase in the outflow facility and PEFL.
The human eye does not exhibit washout effects.17,25 Thus, this two-color tracer perfusion technique we have developed can in the future be used to study how the effective filtration area is involved in the generation of outflow resistance in normal human eyes and increased outflow resistance in POAG eyes.
We are grateful for technical assistance provided by Mrs Rozanne Richman.
This work was supported by American Health Assistance Foundation Grant #G2007-013, NIH-EY018712 and the Massachusetts Lions Eye Research Fund to Boston University.
ZHU Jing-yin, Department of Ophthalmology, Boston University School of Medicine, Boston, MA 02118, USA, Department of Ophthalmology, Huashan Hospital of Fudan University, Shanghai 200040, China, Department of Ophthalmology, Huadong Hospital of Fudan University, Shanghai 200040, China.
YE Wen, Department of Ophthalmology, Huashan Hospital of Fudan University, Shanghai 200040, China.
GONG Hai-yan, Department of Ophthalmology, Boston University School of Medicine, Boston, MA 02118, USA.