Effects of Latrunculin-B on Outflow Facility and Trabecular Meshwork Structure in Human Eyes

Abstract

purpose. To determine the effect of the F-actin-disrupting agent latrunculin-B on aqueous outflow facility and trabecular meshwork architecture in human eyes.

methods. After baseline facility measurement in human eye bank eyes (n = 9 pairs), one eye of each pair received anterior chamber exchange and continued perfusion with medium containing 1 μM latrunculin-B. Contralateral eyes were treated in a similar manner with vehicle. Eyes were fixed by anterior chamber exchange and perfusion with universal fixative at 8 mm Hg (corresponding to a physiologic pressure of 15 mm Hg in vivo), and outflow pathway tissues were examined by transmission and scanning electron microscopy.

results. Perfusion of eyes with 1 μM latrunculin-B caused a continuous and ongoing increase in outflow facility, resulting in a net facility difference of 64% 2 hours after drug administration (P < 0.006). Transmission electron microscopy showed subtle and focal detachment of the inner wall of Schlemm’s canal, rarefaction of the juxtacanalicular tissue (JCT), and cell-cell and cell-matrix detachment. Scanning electron microscopy showed collapsed vacuoles in the inner wall of Schlemm’s canal and a marked increase in the number and size of border (paracellular) pores in the inner wall.

conclusions. Latrunculin-B increases outflow facility in postmortem human eyes. The mechanism of facility increase is most likely due to loss of mechanical integrity of the trabecular meshwork as a consequence of reduction in cell-cell and cell-matrix adhesion. The facility increase and the extent of inner wall separation from the JCT that we observed were both qualitatively similar to that reported in living monkey eyes, but the magnitude of the facility increase and morphologic changes were much less than in the living monkey. This supports the idea that inner wall separation from the JCT may modulate outflow facility.

Numerous studies have shown that agents that alter cytoskeletal function in trabecular meshwork cells also increase outflow facility (for review, see Ref. ¹ ). Agents that directly or indirectly disrupt F-actin, such as H-7, ² the latrunculins, ^{3 4} and the cytochalasins, are particularly efficacious in this regard. The precise mechanism by which such agents increase outflow facility is not definitively known. 

The latrunculins are specific actin-disrupting agents that act by sequestering monomeric G-actin, thereby promoting depolymerization of F-actin filaments. ^{5 6} Among the latrunculins, latrunculin (Lat)-B has been the most extensively tested for its effects on outflow facility. It increases outflow facility in living monkey eyes at high ^{3 7} and low ⁸ doses, as well as in perfused porcine anterior segments. ⁹ However, the effects of Lat-B on outflow facility have not been tested in human eyes. 

The goals of the present work were twofold. First, we wanted to determine the effects of Lat-B on outflow facility in enucleated human eyes. Second, we wanted to investigate how Lat-B affects ultrastructure and architecture of the trabecular meshwork (including the inner wall of Schlemm’s canal) so as to gain insight into the mechanism(s) whereby Lat-B affects outflow facility. 

Our results show that perfusion with 1 μM Lat-B has a dramatic influence on outflow facility in enucleated human eyes, similar in magnitude to that in enucleated porcine eyes, but less than that in living monkey eyes. However, unlike in the monkey eye, this facility increase is accompanied by only very modest rarefaction of the juxtacanalicular tissue (JCT) and separation of the inner wall of Schlemm’s canal from the JCT. However, there were increased openings between inner wall cells (more border or paracellular pores) in Lat-B-treated human eyes. 

Methods

Perfusion and Facility Measurements

All perfusions used Dulbecco’s phosphate-buffered saline with 5.5 mM glucose added (DBG) as mock aqueous humor. DBG fluid was prefiltered through a 0.22-μm filter (Millex-GS; Millipore, Bedford, MA) before use. Lat-B was obtained from Calbiochem (La Jolla, CA) and a 2 mM stock solution was made by dissolving the Lat-B in dimethyl sulfoxide (DMSO), which was aliquotted and frozen. Working solutions (1 μM) were obtained by diluting the stock solutions with DBG. Control solutions contained an equivalent amount of DMSO (0.05%). 

Ostensibly normal human eyes were obtained from eye banks and managed in accordance with the Declaration of Helsinki. After discarding four pairs of eyes with unstable or asymmetric baseline facility traces, or unacceptable trabecular meshwork morphology, nine pairs remained in the study. The mean donor age was 76.1 years (range, 64–83), and the mean postmortem time to the start of perfusion was 22.5 hours (range, 16–27.5). Baseline facility was measured for 60 to 90 minutes by perfusion into the posterior chamber with DBG. All perfusions were performed at a constant pressure of 8 mm Hg (corresponding to 15 mm Hg in vivo) using previously described techniques. ¹⁰ At the completion of baseline facility measurement, one eye of each pair received anterior chamber exchange with a solution of 1 μM Lat-B. Contralateral eyes were treated in a similar manner with vehicle. Care was taken during the anterior chamber exchange to maintain a constant IOP of 8 mm Hg. Eyes were then further perfused with their respective solution at 8 mm Hg for approximately 120 minutes, while measuring facility. 

One of these nine pairs of eyes was noteworthy. It was originally identified as being from a donor with primary open-angle glaucoma. A review of the medical records indicated that the donor, an 89-year-old woman, had received a diagnosis of primary open-angle glaucoma in the mid-1980s and was treated with betaxolol HCl and pilocarpine at that time. Cataract surgery was performed in 1993 (OS) and 1998 (OD) and glaucoma medications were discontinued in 1993. IOP measurements 1 month before death in 2001 were 14 mm Hg OU, and facility measurements in our laboratory gave baseline facilities of 0.20 μL/min per mm Hg OU. We therefore considered these eyes to be normal, but because of their unusual history (including 8 years of glaucoma medications) we examined how they might differ from the other eyes in the study, as described more fully later. 

We present the facility data in two ways. The first is obtained by simple averaging of facility data between eyes, and the results are reported as the mean ± SEM. The second is obtained by normalizing the raw facility data, thereby allowing comparison of relative facility changes between pairs of eyes. Normalization involved dividing the measured facility at each time for a given eye by the average facility reading for that eye in the 30-minute period before anterior chamber exchange with Lat-B. The percentage facility change due to Lat-B perfusion was then computed as 100 · (C _{norm, post} − C _{norm, pre}), where C _{norm, pre} and C _{norm, post} were the normalized facilities before and after Lat-B exchange. The statistical significance of facility changes due to Lat-B was computed from a paired two-tailed Student’s t-test using the percentage normalized facility change as the statistic of interest. 

Morphology

At the conclusion of facility measurement after Lat-B perfusion, all eyes were fixed by anterior chamber exchange and perfusion with 3% paraformaldehyde at 8 mm Hg, after which the eye was opened, and wedges of outflow tissue were cut. Half the wedges from each eye, including tissue from every quadrant, were then immersion fixed overnight in universal fixative (2.5% paraformaldehyde, 2.5% glutaraldehyde in Sörensen’s buffer) for use in electron microscopy, whereas the other half of the wedges were immersion fixed in 3% paraformaldehyde overnight. In two of the nine pairs, the duration of perfusion with fixative was for 60 to 90 minutes. In the other seven pairs, the fixation duration was adjusted so as to equalize fixative volume between fellow eyes of a pair and hence compensate for fixative-induced pore formation in the inner wall of Schlemm’s canal. ¹¹ We refer to this fixation approach as “fixative volume compensation.” 

Tissue from eyes that had been fixed using fixative volume compensation was processed for transmission electron microscopy (TEM) and scanning electron microscopy. Briefly, for TEM, radial segments of the limbal area were dissected, postfixed in 1% osmium tetroxide, dehydrated, infiltrated, and embedded in Epon-Araldite. Ultrathin sections were stained with uranyl acetate and lead citrate. Samples from all four quadrants were examined, except for three pairs of eyes where only two of the four quadrants were examined. Adjacent tissue samples were microdissected and processed using conventional methods ^{12 13} to produce scanning electron microscopy montages of the inner and outer walls of Schlemm’s canal. Standard techniques ^{11 12 14} were used to measure the density and size of pores between inner wall cells (paracellular, border or “B ” pores) and pores through inner wall cells (intracellular or “I ” pores). Inner wall samples from all four quadrants were analyzed in four pairs of eyes and from one quadrant only in two pairs of eyes. The average area analyzed per quadrant by SEM was 128,000 μm². 

Results

Facility Effects of Lat-B

Perfusion with 1 μM Lat-B had a rapid and significant effect on outflow facility (Fig. 1 , Table 1 ), with facility increasing in the treated eye as soon as perfusion measurements could be recommenced after the anterior chamber exchange. Facility increased gradually during the entire 2-hour period after exchange, with no signs of leveling off. There was a small increase in outflow facility in the control eyes which was not statistically significant in the 90- to 120-minute postexchange period. The net difference in normalized facility between treated and control eyes during the period 90 to 120 minutes after exchange was 64%, which was statistically significant (P = 0.006). Fixation decreased facility by approximately 65% in both control and experimental eyes when compared with facility immediately before fixation. When the data were reanalyzed omitting the ostensibly normal pair that had received 8 years of glaucoma medication, almost identical results were obtained, with a net increase in normalized facility of 66% instead of 64%. 

Morphologic Findings in Lat-B-Perfused Eyes

By transmission electron microscopy, there were several consistent differences observed between treated and control eyes. In treated eyes, there was occasional focal detachment of the inner wall of Schlemm’s canal from the underlying JCT (compare Fig. 2with Fig. 3 ). In such regions, and occasionally elsewhere, cellular processes associated with cell-cell and cell-matrix attachments were attenuated and thinned. Focal rarefaction of the JCT was also observed. These observations are qualitatively similar to those reported in the outflow tract of Lat-B-perfused monkey eyes. ¹⁵ However, the extent and magnitude of the changes were much less in the human eyes than those reported in monkey eyes (see, for example, Figs. 3a 3c and 3d and Fig. 6bof Sabanay et al. ¹⁵ ) In fact, many regions of the inner wall and JCT of treated eyes looked essentially normal (Fig. 4) , and we were not always able to tell treated from control eyes when single quadrants were examined in a masked manner. No notable differences were seen between the ostensibly normal pair that had received glaucoma medications and the other pairs of eyes. 

Transmission electron microscopy also showed occasional vacuoles that appeared collapsed or deflated, with a crenellated cellular margin. This observation was consistent with scanning electron microscopic observation of the inner wall of Schlemm’s canal, which showed regions of collapsed and flattened vacuoles (Figs. 5 6) . By scanning electron microscopy, the inner wall of Schlemm’s canal in most of the treated eyes showed more pores, especially openings between adjacent inner wall cells (B pores or paracellular pores). The exception to this trend was in the pair of eyes that had been treated with glaucoma medications, which were very unusual in several ways. Specifically, they showed (1) a higher density of paracellular pores in the control eye than in other control eyes (728 pores/mm² versus an average of 342 pores/mm² in all other control eyes from this study); (2) a 49% decrease in paracellular pore density (Lat-B eye versus control eye), which was opposite to that seen in all other eyes; and (3) a 32% decrease in the size of the B pores (Lat-B eye versus control eye), which was also opposite to the trend seen in all other eyes. For purposes of inner wall pore counts, we therefore treated this pair of eyes as an outlier and neglected them for quantitative analysis of the inner wall of Schlemm’s canal. 

Quantitative analysis of all eyes except the pair that had been treated with glaucoma medications showed a mean increase in B pore density of 247% (range, 63%–476%) due to Lat-B treatment. The increase in paracellular (border) pore density was evident for almost all pore sizes, but was particularly pronounced for the larger pores (Fig. 7) . The net effect was a significant increase in mean pore size (D) as well as pore density (n), and a very pronounced increase in the product of these two quantities (Table 2) . Of note, there was very little effect on the density or size of intracellular (I) pores, with a small decrease in I pore density in treated eyes (not statistically significant) and a small increase in I pore size in treated eyes. No statistically significant correlations were found when percentage facility change was regressed on inner wall pore statistics (B pore density, nD product for B pores, total pore density, nD product for total pores). 

Discussion

The major conclusion of the present work is that 1 μM Lat-B increases outflow facility when perfused into enucleated human postmortem eyes. The facility increase that we observed after Lat-B infusion was similar in character to that reported in living monkey ^{3 15} and organ-cultured porcine ⁹ eyes, showing a gradual increase over several hours. However, the magnitude of the facility increase that we saw was much less than that seen in monkey eyes (447% mean increase in the monkey eye for 2 μM Lat-B 60–90 minutes after drug administration), although comparable to that seen in organ-cultured porcine eyes. This may be a species difference, or a difference between in vivo and postmortem perfusions. 

We also observed a small increase in the facility of control eyes, but this was not statistically significant for the 30-minute period before fixation. This small increase could be due to the DMSO in the vehicle, although, at the concentrations that we used, DMSO has been reported to have no effect on facility in the living monkey eye. ¹⁶ Perhaps human donor eyes are more sensitive to DMSO, which could explain the cell debris that was occasionally seen in the outflow tissues in this study. 

The morphologic correlates of this increased facility included occasional focal detachment of the inner wall of Schlemm’s canal from the JCT and a very pronounced increase in the density and mean size of paracellular pores. We did not measure the extent of inner wall detachment, but estimate that detached areas represented no more than approximately 10% to 15% of the total length of the inner wall of Schlemm’s canal. The inner wall detachment and associated rarefaction of the JCT that we observed is qualitatively consistent with those reported by Sabanay et al. ¹⁵ in the living monkey eye. However, they reported very extensive regions of inner wall detachment, “stretching” of inner wall cells, and associated rarefaction and “ballooning” of the underlying JCT. The inner wall detachment that we observed was much smaller in magnitude and extent than that seen in monkey eyes. There are several possible explanations as to why there was such a pronounced difference between monkey and human eyes in their morphologic and facility responses to Lat-B. It could be a postmortem effect, since we perfused enucleated human eyes while monkey experiments were performed in live animals. It could be an age difference, because the human eyes were relatively elderly while monkeys would tend to be younger. However, we believe that it is most likely that this difference is related to differences in the extent of attachment between the inner wall and the underlying JCT. The human eye is invested with a very extensive network of elastic tendons that extend into the subendothelial region of the JCT, ^{17 18} and these tendons show increased amounts of surrounding sheath-derived plaque materials with age. ¹⁹ This network would be likely to help stabilize the JCT and inner wall region in the aged human eye, most probably to a greater extent than in younger monkey eyes. The difference between the monkey and human results may also be due in part to differences in the perfusion protocol. When perfusing monkey eyes, it is necessary to elevate IOP above the spontaneous level, thereby leading to increased mechanical stresses on the TM. When the TM is exposed to both cytoskeletal-disrupting agents, such as Lat-B, and elevated mechanical stresses, distension, and ballooning of the JCT may be the natural result. 

The increased inner wall separation seen in the monkey eye seems to be consistent with the greater Lat-B induced facility increase in the monkey eye compared with the human eye. This is also consistent with findings suggesting that washout (present in the monkey and absent in the human ²⁰ ) correlates with inner wall separation from the JCT. ²¹ In addition to inner wall detachment, we observed “collapsed” inner wall giant vacuoles, thinning of the walls of giant vacuoles, and a notable increase in number and size of paracellular pores in Lat-B-treated eyes. Giant vacuole collapse could reflect redirection of fluid to lower resistance pathways (e.g., possibly due to focal JCT rarefaction or increased density of inner wall openings, or possibly may indicate that actomyosin tone is necessary to deflate giant vacuoles. An increased density of inner wall pores was not reported in previous studies of Lat-B, ¹⁵ but could easily have been present but undetectable by transmission and light microscopy of conventional sagittal sections. We estimate that conventional ultrathin sagittal sections through the TM (two per quadrant × four quadrants) visualize only approximately 0.01% of the inner wall, making it difficult to detect even relatively large increases in small, infrequently occurring structures such as inner wall pores. Our experience is that observation of an increase in the density of such structures requires the large sampling area provided by scanning electron microscopy. Of course, because the physiological role of inner wall pores is unclear (including which pores, if any, are artifactual ^{11 22} ), their role in facilitating outflow after lat-B treatment is circumstantial at present. 

As an aside, it is interesting to comment on the features of the pair of eyes that had received glaucoma medications for many years. The facility of these eyes was normal, but the control eye of this pair had almost double the paracellular pore density and size of the other control eyes, and the response of inner wall pores to Lat-B was remarkably different from that in all other eyes. We cannot explain this difference, but note that Grierson et al. ²³ have shown that pilocarpine administration increases inner wall pore size and density. We speculate that pilocarpine administration (or other glaucoma medications) may have affected inner wall pores in this pair of eyes as well. 

Generally speaking, the morphologic changes we observed were consistent with a loss of mechanical integrity in the trabecular meshwork/inner wall of Schlemm’s canal. Considering the rapidity and the magnitude of the Lat-B induced facility increase, this loss of integrity due to reduced cell-cell and cell-matrix attachment seems to be the most likely source of the facility change. We cannot say what the ultimate cause of the facility increase was. Likely candidates include the increase in inner wall paracellular pore density and inner wall separation from the JCT. However, the fact that greater inner wall separation was associated with greater facility increase (when comparing monkey eyes to human eyes) surely is interesting, is consistent with observations in bovine eyes, ²¹ and suggests that agents that target inner wall-JCT attachment could be important in modulating outflow facility. 

 Presented at the annual Meeting of the Association of Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April 2004.

 Supported by Canadian Institutes of Health Research Grant MA-10051.

 Submitted for publication March 14, 2005; revised August 24 and September 30, 2005; accepted February 27, 2006.

 Disclosure: C.R. Ethier, None; A.T. Read, None; D.W.-H. Chan, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Plot of normalized facility versus time, averaged over all pairs of eyes (n = 9 pairs). Time 0 is taken as the start of anterior chamber exchange for Lat-B (1 μM) infusion. Lines: the mean; error bars, SEM at each time point. Transient facility dips and spikes after exchange are due to re-establishment of steady state perfusion conditions after anterior chamber exchange and should be ignored in favor of later values.

Table 1.

 

Summary of Facility Data in Lat-B Perfusions

Perfusion Protocol	Facility (μL/min/mmHg)
		Baseline	After Exchange	After Fixation
Control	0.242 ± 0.023	0.304 ± 0.046	0.209 ± 0.051
1 μM Lat-B	0.250 ± 0.035	0.452 ± 0.038	0.332 ± 0.041

 Facility is expressed as mean microliters per minute per mm Hg ± SEM. Baseline facility was obtained by averaging over a 30-minute period before Lat-B exchange; After Exchange facility was obtained by averaging over a 30-minute period before fixative exchange but after Lat-B (or vehicle) exchange (n = 9 pairs); after fixation facility was obtained by averaging over a 30-minute period after fixative exchange.

Figure 2.

 

Transmission electron micrographs of outflow tissue from a control eye. Left: overview of outer TM, JCT, and Schlemm’s canal (SC) tissue. Note the partially collapsed vacuole in the bottom portion of the micrograph. (A, inset) Apparently normal cell-cell and cell-matrix attachment, as well as normally inflated giant vacuoles. This is the contralateral eye to that shown in Figure 3 . Facility data for this eye are shown with the thin line in inset region (D) of Figure 3 .

Figure 3.

 

Transmission electron micrographs of outflow pathway tissue from Lat-B-perfused eye. Top left: overview of outer TM, JCT, and Schlemm’s canal (SC), including a partially collapsed vacuole ( ). Inset region (A) overlies a rarefied JCT zone, but shows good connections (C) between inner wall and underlying JCT extracellular matrix. Inset region (B) shows focal inner wall separation, including apparent sites of detachment between inner wall cells and JCT matrix materials (D). Inset region (C) shows relatively normal cell-cell and cell-matrix attachments. The portion of the cell forming the giant vacuole shows focal thinning. Some cell debris is visible (e.g., in the middle of inset region C). Facility traces for this eye are shown as the thick line in inset region (D).

Figure 6.

 

Scanning electron micrograph of Schlemm’s canal inner wall in an Lat-B-treated eye. Note that many of the giant vacuoles have collapsed, giving a flattened appearance to the inner wall, and that inner wall B pores appear to be more numerous. Arrows identify selected B pores and arrowheads identify selected I pores. This image was taken from the eye depicted in Figure 3(contralateral eye to that shown in Fig. 5 ; see legend).

Figure 4.

 

Transmission electron micrographs of outflow pathway tissue from Lat-B-perfused eye, in this case demonstrating nearly normal TM morphology (compare with more severely affected eye shown in Fig. 3 ). Bottom left: shows overview of outer TM, JCT, and Schlemm’s canal (SC), including a partially collapsed vacuole ( ). Inset regions (A) and (B) show slightly elongated connections (C) between JCT and the outermost trabecular beams, with one apparent site of cell-cell detachment (D). The JCT does not appear to be rarefied in these regions. Inset region (C) shows relatively normal cell-cell and cell-matrix attachments, with no notable tissue rarefaction.

Figure 5.

 

Scanning electron micrograph of Schlemm’s canal inner wall from a control eye. Note elongated endothelial cells characterized by well-developed giant vacuoles, some of which are partially collapsed ( ). Inner wall pores are evident, with arrows identifying selected B pores and arrowheads identifying selected I pores. The remains of a septum are visible in the center of the picture. This image was from the eye depicted in Figure 2 .

Figure 7.

 

Histogram of intercellular (B or paracellular) pores in treated and control eyes. Data are the mean ± SEM for each category. *Statistically significant difference between treated and control eyes (P < 0.05). This histogram omits the data from the single pair of eyes that had been treated with glaucoma medications.

Table 2.

 

Summary of Inner Wall Pore Characteristics in Perfused Eyes

	Control Eyes	Lat-B Treated Eyes	P Value
Border pores
Pore density, n (pores/mm2)	342 ± 110	857 ± 127	0.01
Pore size, D (μm)	1.36 ± 0.15	1.72 ± 0.17	0.08
nD product	439 ± 119	1578 ± 343	0.02
Intracellular pores
Pore density, n (pores/mm2)	416 ± 85	392 ± 45	0.85
Pore size, D (μm)	0.89 ± 0.11	1.01 ± 0.05	0.49
nD product	331 ± 50	391 ± 42	0.47
Total pores
Pore density, n (pores/mm2)	760 ± 182	1256 ± 136	0.05
Pore size, D (μm)	1.07 ± 0.08	1.51 ± 0.15	0.08
nD product	773 ± 161	1977 ± 346	0.02

 Data are the mean ± SEM. Total pore density does not equal the sum of border and intracellular pores due to a small number of pores that could not be classified as either type. This table includes only eyes perfused with fixative volume compensation and excludes the pair of eyes treated with glaucoma medication. nD product is in units of μm/mm². P refers to paired comparison between control and Lat-B treated eyes.

The authors thank Meenal Agarwal for valuable assistance in pore counts; Douglas Johnson for providing comments on the transmission electron micrographs; and the donors’ families and the staff at the Eye Bank of Canada (Ontario Division) and the National Disease Research Interchange (NDRI; Philadelphia, PA) for providing tissue.