Individual astrocyte morphology in the collagenous lamina cribrosa revealed by multicolor DiOlistic labeling.

Astrocytes in the lamina region of the optic nerve head play vital roles in supporting retinal ganglion cell axon health. In glaucoma, these astrocytes are implicated as early responders to stressors, undergoing characteristic changes in cell function as well as cell morphology. Much of what is currently known about individual lamina astrocyte morphology has been learned from rodent models which lack a defining feature of the human optic nerve head, the collagenous lamina cribrosa (LC). Current methods available for evaluation of collagenous LC astrocyte morphology have significant shortcomings. We aimed to evaluate Multicolor DiOlistic labeling (MuDi) as an approach to reveal individual astrocyte morphologies across the collagenous LC. Gold microcarriers were coated with all combinations of three fluorescent cell membrane dyes, DiI, DiD, and DiO, for a total of seven dye combinations. Microcarriers were delivered to 150 µm-thick coronal vibratome slices through the LC of pig, sheep, goat, and monkey eyes via MuDi. Labeled tissues were imaged with confocal and second harmonic generation microscopy to visualize dyed cells and LC collagenous beams, respectively. GFAP labeling of DiOlistically-labeled cells with astrocyte morphologies was used to investigate cell identity. 3D models of astrocytes were created from confocal image stacks for quantification of morphological features. DiOlistic labeling revealed fine details of LC astrocyte morphologies including somas, primary branches, higher-order branches, and end-feet. Labeled cells with astrocyte morphologies were GFAP+. Astrocytes were visible across seven distinct color channels, allowing high labeling density while still distinguishing individual cells from their neighbors. MuDi was capable of revealing tens to hundreds of collagenous LC astrocytes, in situ, with a single application. 3D astrocyte models allowed automated quantification of morphological features including branch number, length, thickness, hierarchy, and straightness as well as Sholl analysis. MuDi labeling provides an opportunity to investigate morphologies of collagenous LC astrocytes, providing both qualitative and quantitative detail, in healthy tissues. This approach may open doors for research of glaucoma, where astrocyte morphological alterations are thought to coincide with key functional changes related to disease progression.

Tissue-engineered anterior segment eye cultures demonstrate hallmarks of conventional organ culture.

BACKGROUND: Glaucoma is a blinding disease largely caused by dysregulation of outflow through the trabecular meshwork (TM), resulting in elevated intraocular pressure (IOP). We hypothesized that transplanting TM cells into a decellularized, tissue-engineered anterior segment eye culture could restore the outflow structure and function. METHODS: Porcine eyes were decellularized with freeze-thaw cycles and perfusion of surfactant. We seeded control scaffolds with CrFK cells transduced with lentiviral vectors to stably express eGFP and compared them to scaffolds seeded with primary TM cells as well as to normal, unaltered eyes. We tracked the repopulation behavior, performed IOP maintenance challenges, and analyzed the histology. RESULTS: Transplanted cells localized to the TM and progressively infiltrated the extracellular matrix, reaching a distribution comparable to normal, unaltered eyes. After a perfusion rate challenge to mimic a glaucomatous pressure elevation, transplanted and normal eyes reestablished a normal intraocular pressure (transplanted = 16.5 ± 0.9 mmHg, normal = 16.9 ± 0.9). However, eyes reseeded with eGFP-expressing CrFK cells could not regulate IOP, remaining high and unstable (27.0 ± 6.2 mmHg) instead. CONCLUSION: Tissue-engineered anterior segment scaffolds can serve as readily available, scalable ocular perfusion cultures. This could reduce dependency on scarce donor globes in outflow research and may allow engineering perfusion cultures with specific geno- and phenotypes.

Real-time imaging of optic nerve head collagen microstructure and biomechanics using instant polarized light microscopy.

Current tools lack the temporal or spatial resolution necessary to image many important aspects of the architecture and dynamics of the optic nerve head (ONH). We evaluated the potential of instant polarized light microscopy (IPOL) to overcome these limitations by leveraging the ability to capture collagen fiber orientation and density in a single image. Coronal sections through the ONH of fresh normal sheep eyes were imaged using IPOL while they were stretched using custom uniaxial or biaxial micro-stretch devices. IPOL allows identifying ONH collagen architectural details, such as fiber interweaving and crimp, and has high temporal resolution, limited only by the frame rate of the camera. Local collagen fiber orientations and deformations were quantified using color analysis and image tracking techniques. We quantified stretch-induced collagen uncrimping of lamina cribrosa (LC) and peripapillary sclera (PPS), and changes in LC pore size (area) and shape (convexity and aspect ratio). The simultaneous high spatial and temporal resolutions of IPOL revealed complex ONH biomechanics: i) stretch-induced local deformation of the PPS was nonlinear and nonaffine. ii) under load the crimped collagen fibers in the PPS and LC straightened, without torsion and with only small rotations. iii) stretch-induced LC pore deformation was anisotropic and heterogeneous among pores. Overall, with stretch the pores were became larger, more convex, and more circular. We have demonstrated that IPOL reveals details of collagen morphology and mechanics under dynamic loading previously out of reach. IPOL can detect stretch-induced collagen uncrimping and other elements of the tissue nonlinear mechanical behavior. IPOL showed changes in pore morphology and collagen architecture that will help improve understanding of how LC tissue responds to load.

Lamina cribrosa vessel and collagen beam networks are distinct.

Our goal was to analyze the spatial interrelation between vascular and collagen networks in the lamina cribrosa (LC). Specifically, we quantified the percentages of collagen beams with/without vessels and of vessels inside/outside of collagen beams. To do this, the vasculature of six normal monkey eyes was labeled by perfusion post-mortem. After enucleation, coronal cryosections through the LC were imaged using fluorescence and polarized light microscopy to visualize the blood vessels and collagen beams, respectively. The images were registered to form 3D volumes. Beams and vessels were segmented, and their spatial interrelationship was quantified in 3D. We found that 22% of the beams contained a vessel (range 14%-32%), and 21% of vessels were outside beams (13%-36%). Stated differently, 78% of beams did not contain a vessel (68%-86%), and 79% of vessels were inside a beam (64%-87%). Individual monkeys differed significantly in the fraction of vessels outside beams (p < 0.01 by linear mixed effect analysis), but not in the fraction of beams with vessels (p > 0.05). There were no significant differences between contralateral eyes in the percent of beams with vessels and of vessels outside beams (p > 0.05). Our results show that the vascular and collagenous networks of the LC in monkey are clearly distinct, and the historical notions that each LC beam contains a vessel and all vessels are within beams are inaccurate. We postulate that vessels outside beams may be relatively more vulnerable to mechanical compression by elevated IOP than are vessels shielded inside of beams.

Interplay between intraocular and intracranial pressure effects on the optic nerve head in vivo.

Intracranial pressure (ICP) has been proposed to play an important role in the sensitivity to intraocular pressure (IOP) and susceptibility to glaucoma. However, the in vivo effects of simultaneous, controlled, acute variations in ICP and IOP have not been directly measured. We quantified the deformations of the anterior lamina cribrosa (ALC) and scleral canal at Bruch's membrane opening (BMO) under acute elevation of IOP and/or ICP. Four eyes of three adult monkeys were imaged in vivo with OCT under four pressure conditions: IOP and ICP either at baseline or elevated. The BMO and ALC were reconstructed from manual delineations. From these, we determined canal area at the BMO (BMO area), BMO aspect ratio and planarity, and ALC median depth relative to the BMO plane. To better account for the pressure effects on the imaging, we also measured ALC visibility as a percent of the BMO area. Further, ALC depths were analyzed only in regions where the ALC was visible in all pressure conditions. Bootstrap sampling was used to obtain mean estimates and confidence intervals, which were then used to test for significant effects of IOP and ICP, independently and in interaction. Response to pressure manipulation was highly individualized between eyes, with significant changes detected in a majority of the parameters. Significant interactions between ICP and IOP occurred in all measures, except ALC visibility. On average, ICP elevation expanded BMO area by 0.17 mm2 at baseline IOP, and contracted BMO area by 0.02 mm2 at high IOP. ICP elevation decreased ALC depth by 10 µm at baseline IOP, but increased depth by 7 µm at high IOP. ALC visibility decreased as ICP increased, both at baseline (-10%) and high IOP (-17%). IOP elevation expanded BMO area by 0.04 mm2 at baseline ICP, and contracted BMO area by 0.09 mm2 at high ICP. On average, IOP elevation caused the ALC to displace 3.3 µm anteriorly at baseline ICP, and 22 µm posteriorly at high ICP. ALC visibility improved as IOP increased, both at baseline (5%) and high ICP (8%). In summary, changing IOP or ICP significantly deformed both the scleral canal and the lamina of the monkey ONH, regardless of the other pressure level. There were significant interactions between the effects of IOP and those of ICP on LC depth, BMO area, aspect ratio and planarity. On most eyes, elevating both pressures by the same amount did not cancel out the effects. Altogether our results show that ICP affects sensitivity to IOP, and thus that it can potentially also affect susceptibility to glaucoma.

Role of radially aligned scleral collagen fibers in optic nerve head biomechanics.

Collagen fibers organized circumferentially around the canal in the peripapillary sclera are thought to provide biomechanical support to the sensitive tissues within the optic nerve head (ONH). Recent studies have demonstrated the existence of a family of fibers in the innermost sclera organized radially from the scleral canal. Our goal was to determine the role of these radial fibers in the sensitivity of scleral canal biomechanics to acute increases in intraocular pressure (IOP). Following the same general approach of previous parametric sensitivity studies, we created nonlinear generic finite element models of a posterior pole with various combinations of radial and circumferential fibers at an IOP of 0 mmHg. We then simulated the effects of normal and elevated IOP levels (15 and 30 mmHg). We monitored four IOP-induced geometric changes: peripapillary sclera stretch, scleral canal displacement, lamina cribrosa displacement, and scleral canal expansion. In addition, we examined the radial (maximum tension) and through-thickness (maximum compression) strains within the ONH tissues. Our models predicted that: 1) radial fibers reduced the posterior displacement of the lamina, especially at elevated IOP; 2) radial fibers reduced IOP-induced radial strain within the peripapillary sclera and retinal tissue; and 3) a combination of radial and circumferential fibers maintained strains within the ONH at a level similar to those conferred by circumferential fibers alone. In conclusion, radial fibers provide support for the posterior globe, additional to that provided by circumferential fibers. Most importantly, a combination of both fiber families can better protect ONH tissues from excessive IOP-induced deformation than either alone.

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract.

PURPOSE: To characterize the effects of netarsudil on the aqueous humor outflow tract distal to the trabecular meshwork (TM). We hypothesized that netarsudil increases outflow facility in eyes with and without circumferential ab interno trabeculectomy (AIT) that removes the TM. METHODS: Sixty-four porcine anterior segment cultures were randomly assigned to groups with (n = 32) and without circumferential AIT (n = 32). Cultures were exposed to 0.1, 1, and 10 µM netarsudil (N = 8 eyes per concentration). For each concentration, IOP and vessel diameters were compared with their respective pretreatment baselines. Outflow tract vessel diameters were assessed by spectral-domain optical coherence tomography (SDOCT) and rendered in 4D (XYZ time series). RESULTS: Netarsudil at 1 µM reduced IOP both in eyes with TM (- 0.60 ± 0.24 mmHg, p = 0.01) and in eyes without TM (- 1.79 ± 0.42 mmHg, p < 0.01). At this concentration, vessels of the distal outflow tract dilated by 72%. However, at 0.1 µM netarsudil elevated IOP in eyes with TM (1.59 ± 0.36 mmHg, p < 0.001) as well as in eyes without TM (0.23 ± 0.32 mmHg, p < 0.001). Vessels of the distal outflow tract constricted by 31%. Similarly, netarsudil at a concentration of 10 µM elevated IOP both in eyes with TM (1.91 ± 0.193, p < 0.001) and in eyes without TM (3.65 ± 0.86 mmHg, p < 0.001). At this concentration, outflow tract vessels constricted by 27%. CONCLUSION: In the porcine anterior segment culture, the dose-dependent IOP changes caused by netarsudil matched the diameter changes of distal outflow tract vessels. Hyper- and hypotensive properties of netarsudil persisted after TM removal.

RKI-1447, a Rho kinase inhibitor, causes ocular hypotension, actin stress fiber disruption, and increased phagocytosis.

PURPOSE: This study investigated the hypotensive effect of RKI-1447, a Rho kinase inhibitor, in a porcine ex vivo pigmentary glaucoma model. METHODS: Twenty-eight porcine anterior chambers were perfused with medium supplemented with 1.67 × 107 pigment particles/ml for 48 h before treatment with RKI-1447 (n = 16) or vehicle control (n = 12). Intraocular pressure (IOP) was recorded and outflow facility was calculated. Primary trabecular meshwork cells were exposed to RKI-1447 or vehicle control; effects on the cytoskeleton, motility, and phagocytosis were evaluated. RESULT: Compared to baseline, the perfusion of pigment caused a significant increase in IOP in the RKI-1447 group (P = 0.003) at 48 h. Subsequent treatment with RKI-1447 significantly reduced IOP from 20.14 ± 2.59 to 13.38 ± 0.91 mmHg (P = 0.02). Pigment perfusion reduced the outflow facility from 0.27 ± 0.03 at baseline to 0.18 ± 0.02 at 48 h (P < 0.001). This was partially reversed with RKI-1447. RKI-1447 caused no apparent histological changes in the micro- or macroscopic TM appearance. RKI-1447-treated primary TM cells showed significant disruption of the actin cytoskeleton both in the presence and absence of pigment (P < 0.001) but no effect on TM migration was observed. Pigment-treated TM cells exhibited a reduction in TM phagocytosis, which RKI-1447 reversed. CONCLUSION: RKI-1447 significantly reduces IOP by disrupting TM stress fibers and increasing TM phagocytosis. These features may make it useful for the treatment of secondary glaucomas with an increased phagocytic load.

Impact of pigment dispersion on trabecular meshwork cells.

PURPOSE: Dysfunction of the trabecular meshwork (TM) in pigmentary glaucoma contributes to increased aqueous humor outflow resistance and intraocular pressure. In this study, we investigated the effect of pigment dispersion on trabecular meshwork cells. METHODS: Porcine TM cells from ab interno trabeculectomy specimens were exposed to pigment dispersion, then, analyzed for changes in morphology, immunostaining, and ultrastructure. Their abilities to phagocytose migrate, and contraction was quantified. An expression microarray, using 23,937 probes, and a pathway analysis were performed. RESULTS: Stress fiber formation was increased in the pigment dispersion group (P) (60.1 ± 0.3%, n = 10) compared to control (C) (38.4 ± 2.5%, n = 11, p < 0.001). Phagocytosis declined (number of cells with microspheres in P = 37.0 ± 1.1% and in C = 68.7 ± 1.3%, n = 3, p < 0.001) and migration was reduced after 6 h (cells within the visual field over 6 h in P = 28.0.1 ± 2.3 (n = 12) and in C = 40.6 ± 3.3 (n = 13), p < 0.01). Pigment induced contraction at 24 h onwards (p < 0.01). Microarray analysis revealed that Rho signaling was central to these responses. CONCLUSION: Exposure of TM cells to pigment dispersion resulted in reduced phagocytosis and migration, as well as increased stress fiber formation and cell contraction. The Rho signaling pathway played a central and early role, suggesting that its inhibitors could be used as a specific intervention in treatment of pigmentary glaucoma.

Outflow facility and extent of angle closure in a porcine model.

PURPOSE: To establish the extent of anterior chamber angle circumference needed to maintain a physiological outflow facility (C). This could create a model to investigate focal outflow regulation. METHODS: Twenty anterior segments of porcine eyes were assigned to five groups, each with a different degree of cyanoacrylate-mediated angle closure: 90° (n = 4), 180° (n = 4), 270° (n = 4), 360° (n = 4), and four unoccluded control eyes. The outflow facility was measured at baseline, 3, 12, 24, and 36 h after angle closure. Outflow patterns were evaluated with canalograms and the histomorphology was compared. RESULTS: Baseline outflow facilities of the five groups were similar (F = 0.922, p = 0.477). Occlusion of 360° induced a significant decrease in facility from baseline at all time-points (p ≤ 0.023 at 3, 12, 24, and 36 h). However, no difference from baseline was found in any of the partially occluded (0-270°) groups (F ≥ 0.067, p ≥ 0.296 at 3, 12, 24, and 36 h). The canalograms confirmed the extent of occlusion with flow through the unblocked regions. Histology revealed no adverse effects of blockage on the TM or aqueous plexus in the unoccluded angle portions. The unoccluded TM appeared normal. CONCLUSION: Cyanoacrylate-mediated angle occlusion created a reproducible angle closure model. Ninety degrees of unoccluded anterior chamber angle circumference was sufficient to maintain physiological outflow. This model may help understand how outflow can be regulated in healthy, nonglaucomatous TM.

Outflow enhancement by three different ab interno trabeculectomy procedures in a porcine anterior segment model.

PURPOSE: To evaluate three different microincisional ab interno trabeculectomy procedures in a porcine eye perfusion model. METHODS: In perfused porcine anterior segments, 90° of trabecular meshwork (TM) was ablated using the Trabectome (T; n = 8), Goniotome (G; n = 8), or Kahook device (K; n = 8). After 24 h, additional 90° of TM was removed. Intraocular pressure (IOP) and outflow facility were measured at 5 and 10 µl/min perfusion to simulate an elevated IOP. Structure and function were assessed with canalograms and histology. RESULTS: At 5 µl/min infusion rate, T resulted in a greater IOP reduction than G or K from baseline (76.12% decrease versus 48.19% and 47.96%, P = 0.013). IOP reduction between G and K was similar (P = 0.420). Removing another 90° of TM caused an additional IOP reduction only in T and G but not in K. Similarly, T resulted in the largest increase in outflow facility at 5 µl/min compared with G and K (first ablation, 3.41 times increase versus 1.95 and 1.87; second ablation, 4.60 versus 2.50 and 1.74) with similar results at 10 µl/min (first ablation, 3.28 versus 2.29 and 1.90 (P = 0.001); second ablation, 4.10 versus 3.01 and 2.01 (P = 0.001)). Canalograms indicated circumferential flow beyond the ablation endpoints. CONCLUSIONS: T, G, and K significantly increased the outflow facility. In this model, T had a larger effect than G and K.

Morphological comparison of astrocytes in the lamina cribrosa and glial lamina.

Purpose: Although the mechanisms underlying glaucomatous neurodegeneration are not yet well understood, cellular and small animal models suggest that LC astrocytes undergo early morphologic and functional changes, indicating their role as early responders to glaucomatous stress. These models, however, lack the LC found in larger animals and humans, leaving the in situ morphology of LC astrocytes and their role in glaucoma initiation underexplored. In this work, we aimed to characterize the morphology of LC astrocytes in situ and determine differences and similarities with astrocytes in the mouse glial lamina (GL), the analogous structure in a prominent glaucoma model. Methods: Astrocytes in the LCs of twenty-two eyes from goats, sheep, and pigs were stochastically labeled via Multicolor DiOlistics and imaged in situ using confocal microscopy. 3D models of DiOlistically-labeled LC astrocytes and hGFAPpr-GFP mouse GL astrocytes were constructed to quantify morphological features related to astrocyte functions. LC and GL astrocyte cross-pore contacts, branching complexity, branch tortuosity, and cell and branch span were compared. Results: LC astrocytes displayed distinct spatial relationships with collagen, greater branching complexity, and higher branch tortuosity compared to GL astrocytes. Despite substantial differences in their anatomical environments, LC and GL astrocytes had similar cell and branch spans. Conclusions: Astrocyte morphology in the LC was characterized through Multicolor DiOlistic labeling. LC and GL astrocytes have both distinct and shared morphological features. Further research is needed to understand the potentially unique roles of LC astrocytes in glaucoma initiation and progression.

Direct measurements of collagen fiber recruitment in the posterior pole of the eye.

Collagen is the main load-bearing component of the peripapillary sclera (PPS) and lamina cribrosa (LC) in the eye. Whilst it has been shown that uncrimping and recruitment of the PPS and LC collagen fibers underlies the macro-scale nonlinear stiffening of both tissues with increased intraocular pressure (IOP), the uncrimping and recruitment as a function of local stretch have not been directly measured. This knowledge is crucial to understanding their functions in bearing loads and maintaining tissue integrity. In this project we measured local stretch-induced collagen fiber bundle uncrimping and recruitment curves of the PPS and LC. Thin coronal samples of PPS and LC of sheep eyes were mounted and stretched biaxially quasi-statically using a custom system. At each step, we imaged the PPS and LC with instant polarized light microscopy and quantified pixel-level (1.5 µm/pixel) collagen fiber orientations. We used digital image correlation to measure the local stretch and quantified collagen crimp by the circular standard deviation of fiber orientations, or waviness. Local stretch-recruitment curves of PPS and LC approximated sigmoid functions. PPS recruited more fibers than the LC at the low levels of stretch. At 10% stretch the curves crossed with 75% bundles recruited. The PPS had higher uncrimping rate and waviness remaining after recruitment than the LC: 0.9º vs. 0.6º and 3.1º vs. 2.7º. Altogether our findings support describing fiber recruitment of both PPS and LC with sigmoid curves, with the PPS recruiting faster and at lower stretch than the LC, consistent with a stiffer tissue. STATEMENT OF SIGNIFICANCE: Peripapillary sclera (PPS) and lamina cribrosa (LC) collagen recruitment behaviors are central to the nonlinear mechanical behavior of the posterior pole of the eye. How PPS and LC collagen fibers recruit under stretch is crucial to develop constitutive models of the tissues but remains unclear. We used image-based stretch testing to characterize PPS and LC collagen fiber bundle recruitment under local stretch. We found that fiber-level stretch-recruitment curves of PPS and LC approximated sigmoid functions. PPS recruited more fibers at a low stretch, but at 10% bundle stretch the two curves crossed with 75% bundles recruited. We also found that PPS and LC fibers had different uncrimping rates and non-zero waviness's when recruited.

Direct measurements of collagen fiber recruitment in the posterior pole of the eye.

Collagen is the main load-bearing component of the peripapillary sclera (PPS) and lamina cribrosa (LC) in the eye. Whilst it has been shown that uncrimping and recruitment of the PPS and LC collagen fibers underlies the macro-scale nonlinear stiffening of both tissues with increased intraocular pressure (IOP), the uncrimping and recruitment as a function of local stretch have not been directly measured. This knowledge is crucial for the development of constitutive models associating micro and macro scales. In this project we measured local stretch-induced collagen fiber bundle uncrimping and recruitment curves of the PPS and LC. Thin coronal samples of PPS and LC of sheep eyes were mounted and stretched biaxially quasi-statically using a custom system. At each step, we imaged the PPS and LC with instant polarized light microscopy and quantified pixel-level (1.5 µm/pixel) collagen fiber orientations. We used digital image correlation to measure the local stretch and quantified collagen crimp by the circular standard deviation of fiber orientations, or waviness. Local stretch-recruitment curves of PPS and LC approximated sigmoid functions. PPS recruited more fibers than the LC at the low levels of stretch. At 10% stretch the curves crossed with 75% bundles recruited. The PPS had higher uncrimping rate and waviness remaining after recruitment than the LC: 0.9° vs. 0.6° and 3.1° vs. 2.7°. Altogether our findings support describing fiber recruitment of both PPS and LC with sigmoid curves, with the PPS recruiting faster and at lower stretch than the LC, consistent with a stiffer tissue.

In vivo Modulation of Intraocular and Intracranial Pressures Causes Nonlinear and Non-monotonic Deformations of the Lamina Cribrosa and Scleral Canal.

Purpose: To evaluate changes in monkey optic nerve head (ONH) morphology under acutely controlled intraocular pressure (IOP) and intracranial pressure (ICP). Methods: Seven ONHs from six monkeys were imaged via optical coherence tomography while IOP and ICP were maintained at one of 16 conditions. These conditions were defined by 4 levels for each pressure: low, baseline, high and very high. Images were processed to determine scleral canal area, aspect ratio, and planarity and anterior lamina cribrosa (ALC) shape index and curvature. Linear mixed effect models were utilized to investigate the effects of IOP, ICP and their interactions on ONH morphological features. The IOP-ICP interaction model was compared with one based on translaminar pressure difference (TLPD). Results: We observed complex, eye-specific, non-linear patterns of ONH morphological changes with changes in IOP and ICP. For all ONH morphological features, linear mixed effects models demonstrated significant interactions between IOP and ICP that were unaccounted for by TLPD. Interactions indicate that the effects of IOP and ICP depend on the other pressure. The IOP-ICP interaction model was a higher quality predictor of ONH features than a TLPD model. Conclusions: In vivo modulation of IOP and ICP causes nonlinear and non-monotonic changes in monkey ONH morphology that depend on both pressures and is not accounted for by a simplistic TLPD. These results support and extend prior findings. Translational Relevance: A better understanding of ICP's influence on the effects of IOP can help inform the highly variable presentations of glaucoma and effective treatment strategies.

Instant polarized light microscopy for imaging collagen microarchitecture and dynamics.

Collagen fibers are a primary load-bearing component of connective tissues and are therefore central to tissue biomechanics and pathophysiology. Understanding collagen architecture and behavior under dynamic loading requires a quantitative imaging technique with simultaneously high spatial and temporal resolutions. Suitable techniques are thus rare and often inaccessible. In this study, we present instant polarized light microscopy (IPOL), in which a single snapshot image encodes information on fiber orientation and retardance, thus fulfilling the requirement. We utilized both simulation and experimental data from collagenous tissues of chicken tendon, sheep eye, and porcine heart to evaluate the effectiveness of IPOL as a quantitative imaging technique. We demonstrate that IPOL allows quantitative characterization of micron-scale collagen fiber architecture at full camera frame rates (156 frames/second herein).

So-Called Lamina Cribrosa Defects May Mitigate IOP-Induced Neural Tissue Insult.

Purpose: The prevailing theory about the function of lamina cribrosa (LC) connective tissues is that they provide structural support to adjacent neural tissues. Missing connective tissues would compromise this support and therefore are regarded as "LC defects", despite scarce actual evidence of their role. We examined how so-called LC defects alter IOP-related mechanical insult to the LC neural tissues. Methods: We built numerical models incorporating LC microstructure from polarized light microscopy images. To simulate LC defects of varying sizes, individual beams were progressively removed. We then compared intraocular pressure (IOP)-induced neural tissue deformations between models with and without defects. To better understand the consequences of defect development, we also compared neural tissue deformations between models with partial and complete loss of a beam. Results: The maximum stretch of neural tissues decreased non-monotonically with defect size. Maximum stretch in the model with the largest defect decreased by 40% in comparison to the model with no defects. Partial loss of a beam increased the maximum stretch of neural tissues in its adjacent pores by 162%, compared with 63% in the model with complete loss of a beam. Conclusions: Missing LC connective tissues can mitigate IOP-induced neural tissue insult, suggesting that the role of the LC connective tissues is more complex than simply fortifying against IOP. The numerical models further predict that partial loss of a beam is biomechanically considerably worse than complete loss of a beam, perhaps explaining why defects have been reported clinically but partial beams have not.

Ripasudil in a Model of Pigmentary Glaucoma.

Purpose: To investigate the effects of Ripasudil (K-115), a Rho-kinase inhibitor, in a porcine model of pigmentary glaucoma. Methods: In vitro trabecular meshwork (TM) cells and ex vivo perfused eyes were subjected to pigment dispersion followed by K-115 treatment (PK115). PK115 was compared to controls (C) and pigment (P). Cytoskeletal alterations were assessed by F-actin labeling. TM cell phagocytosis of fluorescent targets was evaluated by flow cytometry. Cell migration was studied with a wound-healing assay. Intraocular pressure was continuously monitored and compared to after the establishment of the pigmentary glaucoma model and after treatment with K-115. Results: The percentage of cells with stress fibers increased in response to pigment but declined sharply after treatment with K-115 (P: 32.8% ± 2.9%; PK115: 11.6% ± 3.3%, P < 0.001). Phagocytosis first declined but recovered after K-115 (P: 25.7% ± 2.1%, PK115: 33.4% ± 0.8%, P <0.01). Migration recuperated at 12 hours with K-115 treatment (P: 19.1 ± 4.6 cells/high-power field, PK115: 42.5 ± 1.6 cells/high-power field, P < 0.001). Ex vivo, eyes became hypertensive from pigment dispersion but were normotensive after treatment with K-115 (P: 20.3 ± 1.2 mm Hg, PK115: 8.9 ± 1.7 mm Hg; P < 0.005). Conclusions: In vitro, K-115 reduced TM stress fibers, restored phagocytosis, and restored migration of TM cells. Ex vivo, K-115 normalized intraocular pressure. Translational Relevance: This ex vivo pigmentary glaucoma model provides a readily available basis to investigate new drugs such as the rho-kinase inhibitor studied here.

3D-Reconstruction of the human conventional outflow system by ribbon scanning confocal microscopy.

PURPOSE: The risk for glaucoma is driven by the microanatomy and function of the anterior segment. We performed a computation-intense, high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM) of the outflow tract of two human eyes. We hypothesized this would reveal important species differences when compared to existing data of porcine eyes, an animal that does not spontaneously develop glaucoma. METHODS: After perfusing two human octogenarian eyes with lectin-fluorophore conjugate and optical clearance with benzyl alcohol benzyl benzoate (BABB), anterior segments were scanned by RSCM and reconstructed in 3D for whole-specimen rendering. Morphometric analyses of the outflow tract were performed for the trabecular meshwork (TM), limbal, and perilimbal outflow structures and compared to existing porcine data. RESULTS: RSCM provided high-resolution data for IMARIS-based surface reconstruction of outflow tract structures in 3D. Different from porcine eyes with an abundance of highly interconnected, narrow, and short collector channels (CCs), human eyes demonstrated fewer CCs which had a 1.5x greater cross-sectional area (CSA) and 2.6x greater length. Proximal CC openings at the level of Schlemm's canal (SC) had a 1.3x larger CSA than distal openings into the scleral vascular plexus (SVP). CCs were 10.2x smaller in volume than the receiving SVP vessels. Axenfeld loops, projections of the long ciliary nerve, were also visualized. CONCLUSION: In this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes. Human CCs spanned several clock-hours and were larger than in porcine eyes. These species differences may point to factors downstream of the TM that increase our vulnerability to glaucoma.

Lamina Cribrosa Capillaries Straighten as Intraocular Pressure Increases.

Purpose: The purpose of this study was to visualize the lamina cribrosa (LC) capillaries and collagenous beams, measure capillary tortuosity (path length over straight end-to-end length), and determine if capillary tortuosity changes when intraocular pressure (IOP) increases. Methods: Within 8 hours of sacrifice, 3 pig heads were cannulated via the external ophthalmic artery, perfused with PBS to remove blood, and then perfused with a fluorescent dye to label the capillaries. The posterior pole of each eye was mounted in a custom-made inflation chamber for control of IOP with simultaneous imaging. Capillaries and collagen beams were visualized with structured light illumination enhanced imaging at IOPs from 5 to 50 mm Hg at each 5 mm Hg increment. Capillary tortuosity was measured from the images and paired two-sample t-tests were used to assess for significant changes in relation to changes in IOP. Results: Capillaries were highly tortuous at 15 mm Hg (up to 1.45). In all but one eye, tortuosity decreased significantly as IOP increased from 15 to 25 mm Hg (P < 0.01), and tortuosity decreased significantly in every eye as IOP increased from 15 to 40 mm Hg (P < 0.01). In only 16% of capillaries, tortuosity increased with elevated IOP. Capillaries had a surprisingly different topology from the collagen beams. Conclusions: Although high capillary tortuosity is sometimes regarded as potentially problematic because it can reduce blood flow, LC capillary tortuosity may provide slack that mitigates against reduced flow and structural damage caused by excessive stretch under elevated IOP. We speculate that low capillary tortuosity could be a risk factor for damage under high IOP.