Computational study of the mechanical behavior of the astrocyte network and axonal compartments in the mouse optic nerve head.

Glaucoma is a blinding disease characterized by the degeneration of the retinal ganglion cell (RGC) axons at the optic nerve head (ONH). A major risk factor for glaucoma is the intraocular pressure (IOP). However, it is currently impossible to measure the IOP-induced mechanical response of the axons of the ONH. The objective of this study was to develop a computational modeling method to estimate the IOP-induced strains and stresses in the axonal compartments in the mouse astrocytic lamina (AL) of the ONH, and to investigate the effect of the structural features on the mechanical behavior. We developed experimentally informed finite element (FE) models of six mouse ALs to investigate the effect of structure on the strain responses of the astrocyte network and axonal compartments to pressure elevation. The specimen-specific geometries of the FE models were reconstructed from confocal fluorescent images of cryosections of the mouse AL acquired in a previous study that measured the structural features of the astrocytic processes and axonal compartments. The displacement fields obtained from digital volume correlation in prior inflation tests of the mouse AL were used to determine the displacement boundary conditions of the FE models. We then applied Gaussian process regression to analyze the effects of the structural features on the strain outcomes simulated for the axonal compartments. The axonal compartments experienced, on average, 6 times higher maximum principal strain but 1800 times lower maximum principal stress compared to those experienced by the astrocyte processes. The strains experienced by the axonal compartments were most sensitive to variations in the area of the axonal compartments. Larger axonal compartments that were more vertically aligned, closer to the AL center, and with lower local actin area fraction had higher strains. Understanding the factors affecting the deformation in the axonal compartments will provide insights into mechanisms of glaucomatous axonal damage.

The Curvature, Collagen Network Structure, and Their Relationship to the Pressure-Induced Strain Response of the Human Lamina Cribrosa in Normal and Glaucoma Eyes.

The lamina cribrosa (LC) is a connective tissue in the optic nerve head (ONH). The objective of this study was to measure the curvature and collagen microstructure of the human LC, compare the effects of glaucoma and glaucoma optic nerve damage, and investigate the relationship between the structure and pressure-induced strain response of the LC in glaucoma eyes. Previously, the posterior scleral cups of 10 normal eyes and 16 diagnosed glaucoma eyes were subjected to inflation testing with second harmonic generation (SHG) imaging of the LC and digital volume correlation (DVC) to calculate the strain field. In this study, we applied a custom microstructural analysis algorithm to the maximum intensity projection of SHG images to measure features of the LC beam and pore network. We also estimated the LC curvatures from the anterior surface of the DVC-correlated LC volume. Results showed that the LC in glaucoma eyes had larger curvatures p≤0.03), a smaller average pore area (p = 0.001), greater beam tortuosity (p < 0.0001), and more isotropic beam structure (p = 0.01) than in normal eyes. The difference measured between glaucoma and normal eyes may indicate remodeling of the LC with glaucoma or baseline differences that contribute to the development of glaucomatous axonal damage.

Mechanical strain in the mouse astrocytic lamina increases after exposure to recombinant trypsin.

The responses of astrocytes in the optic nerve head (ONH) to mechanical and biochemical stimuli are important to understanding the degeneration of retinal ganglion cell axons in glaucoma. The ONH in glaucoma is vulnerable to stress produced by the intraocular pressure (IOP). Notably, after three days of elevated IOP in a mouse model, the junctions between the astrocytic processes and the peripapillary sclera were altered and the structural compliance of the ONH increased. In order to simulate this aspect of glaucomatous remodeling, explanted mouse eyes were treated with TrypLE, a recombinant trypsin enzyme. Treatment with TrypLE caused the periphery of the astrocytic lamina to contract radially by 0.044 ± 0.038. Transmission electron microscopy showed that TrypLE caused a separation of the end-feet of the astrocyte processes from the basement membrane at the junction with the sclera. Inflation testing after treatment with TrypLE caused an increased strain response in the astrocytic lamina compared to the strain response before treatment. The greatest increase was in the radial Green-Lagrange strain, Err = 0.028 ± 0.009, which increased by 340%. The alterations in the microstructure and in the strain response of the astrocytic lamina reported in mouse experimental glaucoma were partially reproduced by experimental treatment of mouse eyes with TrypLE. The results herein suggest that separation of junctions between the astrocyte processes and the sclera may be instrumental in increasing the structural compliance of the ONH after a period of elevated IOP. STATEMENT OF SIGNIFICANCE: Astrocytes of the optic nerve of the eye spread out from edge to edge across the optic nerve in a region referred to as the astrocytic lamina. In an experimental model of glaucoma caused by elevated eye-pressure, there is disruption of the connections between astrocytes and the edge of the astrocytic lamina. We caused a similar event in the lamina by incubating explanted mouse eyes with an enzyme. Disruption of the astrocyte connections to the edge of their tissue caused the tissue to stretch more when we increased the eye-pressure, compared to the control tissue. This work is the first on the tissue of the optic nerve to demonstrate the importance of cell connections in preventing the over-stretching of the astrocytic lamina.

Quantitative Microstructural Analysis of Cellular and Tissue Remodeling in Human Glaucoma Optic Nerve Head.

Purpose: To measure quantitatively changes in lamina cribrosa (LC) cell and connective tissue structure in human glaucoma eyes. Methods: We studied 27 glaucoma and 19 age-matched non-glaucoma postmortem eyes. In 25 eyes, LC cross-sections were examined by confocal and multiphoton microscopy to quantify structures identified by anti-glial fibrillary acidic protein (GFAP), phalloidin-labeled F-actin, nuclear 4',6-diamidino-2-phenylindole (DAPI), and by second harmonic generation imaging of LC beams. Additional light and transmission electron microscopy were performed in 21 eyes to confirm features of LC remodeling, including immunolabeling by anti-SOX9 and anti-collagen IV. All glaucoma eyes had detailed clinical histories of open-angle glaucoma status, and degree of axon loss was quantified in retrolaminar optic nerve cross-sections. Results: Within LC pores, the proportionate area of both GFAP and F-actin processes was significantly lower in glaucoma eyes than in controls (P = 0.01). Nuclei were rounder (lower median aspect ratio) in glaucoma specimens (P = 0.02). In models assessing degree of glaucoma damage, F-actin process width was significantly wider in glaucoma eyes with more damage (P = 0.024), average LC beam width decreased with worse glaucoma damage (P = 0.042), and nuclear count per square millimeter rose with worse damage (P = 0.019). The greater cell count in LC pores represented 92.3% astrocytes by SOX9 labeling. The results are consistent with replacement of axons in LC pores by basement membrane labeled by anti-collagen IV and in-migrating astrocytes. Conclusions: Alteration in LC structure in glaucoma involves migration of astrocytes into axonal bundles, change in astrocyte orientation and processes, production of basement membrane material, and thinning of connective tissue beams.

Pressure-Induced Changes in Astrocyte GFAP, Actin, and Nuclear Morphology in Mouse Optic Nerve.

Purpose: To conduct quantitative analysis of astrocytic glial fibrillary acidic protein (GFAP), actin and nuclei distribution in mouse optic nerve (ON) and investigate changes in the measured features after 3 days of ocular hypertension (OHT). Method: Serial cross-sections of 3-day microbead-induced OHT and control ONs were fluorescently labelled and imaged using confocal microscope. Eighteen structural features were measured from the acquired images, including GFAP coverage, actin area fraction, process thickness, and aspect ratio of cell nucleus. The measured features were analyzed for variations with axial locations along ON and radial zones transverse to ON, as well as for the correlations with degree of intraocular pressure (IOP) change. Results: The most significant changes in structural features after 3-day OHT occurred in the unmyelinated ON region (R1), and the changes were greater with greater IOP elevation. Although the GFAP, actin, axonal, and ON areas all increased in 3-day OHT ONs in R1 (P ≤ 0.004 for all), the area fraction of GFAP actually decreased (P = 0.02), the actin area fraction was stable and individual axon compartments were unchanged in size. Within R1, the number of nuclear clusters increased (P < 0.001), but the mean size of nuclear clusters was smaller (P = 0.02) and the clusters became rounder (P < 0.001). In all cross-sections of control ONs, astrocytic processes were thickest in the rim zone compared with the central and peripheral zones (P ≤ 0.002 for both), whereas the overall process width in R1 decreased after 3 days of OHT (P < 0.001). Conclusions: The changes in structure elucidated IOP-generated alterations that underlie astrocyte mechanotranslational responses relevant to glaucoma.

The Effects of Glaucoma on the Pressure-Induced Strain Response of the Human Lamina Cribrosa.

Purpose: To measure the ex vivo pressure-induced strain response of the human optic nerve head and analyze for variations with glaucoma diagnosis and optic nerve axon damage. Methods: The posterior sclera of 16 eyes from 8 diagnosed glaucoma donors and 10 eyes from 6 donors with no history of glaucoma were inflation tested between 5 and 45 mm Hg. The optic nerve from each donor was examined for degree of axon loss. The posterior volume of the lamina cribrosa (LC) was imaged with second harmonic generation and analyzed using volume correlation to calculate LC strains between 5 and 10 and 5 and 45 mm Hg. Results: Eye length and LC area were larger in eyes diagnosed with glaucoma (P ≤ 0.03). Nasal-temporal EXX and circumferential Eθθ strains were lower in the LC of diagnosed glaucoma eyes at 10 mm Hg (P ≤ 0.05) and 45 mm Hg (P ≤ 0.07). EXX was smaller in the LC of glaucoma eyes with <25% axon loss compared with undamaged normal eyes (P = 0.01, 45 mm Hg). In general, the strains were larger in the peripheral than central LC. The ratio of the maximum principal strain Emax in the peripheral to central LC was larger in glaucoma eyes with >25% axon loss than in glaucoma eyes with milder damage (P = 0.004, 10 mm Hg). Conclusions: The stiffness of the LC pressure-strain response was greater in diagnosed glaucoma eyes and varied with glaucomatous axon damage. Lower LC strains in glaucoma eyes with milder damage may represent baseline biomechanical behavior that contributes to axon loss, whereas greater LC strain and altered radial LC strain variation in glaucoma eyes with more severe damage may be caused by glaucoma-related remodeling.

Characterizing the Collagen Network Structure and Pressure-Induced Strains of the Human Lamina Cribrosa.

Purpose: The purpose of this study was to measure the 2D collagen network structure of the human lamina cribrosa (LC), analyze for the correlations with age, region, and LC size, as well as the correlations with pressure-induced strains. Methods: The posterior scleral cups of 10 enucleated human eyes with no known ocular disease were subjected to ex vivo inflation testing from 5 to 45 mm Hg. The optic nerve head was imaged by using second harmonic generation imaging (SHG) to identify the LC collagen structure at both pressures. Displacements and strains were calculated by using digital volume correlation of the SHG volumes. Nine structural features were measured by using a custom Matlab image analysis program, including the pore area fraction, node density, and beam connectivity, tortuosity, and anisotropy. Results: All strain measures increased significantly with higher pore area fraction, and all but the radial-circumferential shear strain (Erθ) decreased with higher node density. The maximum principal strain (Emax) and maximum shear strain (Γmax) also increased with larger beam aspect ratio and tortuosity, respectively, and decreased with higher connectivity. The peripheral regions had lower node density and connectivity, and higher pore area fraction, tortuosity, and strains (except for Erθ) than the central regions. The peripheral nasal region had the lowest Emax, Γmax, radial strain, and pore area fraction. Conclusions: Features of LC beam network microstructure that are indicative of greater collagen density and connectivity are associated with lower pressure-induced LC strain, potentially contributing to resistance to glaucomatous damage.

Deproteinization of Cortical Bone: Effects of Different Treatments.

Bone is a biological composite material having collagen and mineral as its main constituents. In order to better understand the arrangement of the mineral phase in bone, porcine cortical bone was deproteinized using different chemical treatments. This study aims to determine the best method to remove the protein constituent while preserving the mineral component. Chemicals used were H2O2, NaOCl, NaOH, and KOH, and the efficacy of deproteinization treatments was determined by thermogravimetric analysis and Raman spectroscopy. The structure of the residual mineral parts was examined using scanning electron microscopy. X-ray diffraction was used to confirm that the mineral component was not altered by the chemical treatments. NaOCl was found to be the most effective method for deproteinization and the mineral phase was self-standing, supporting the hypothesis that bone is an interpenetrating composite. Thermogravimetric analyses and Raman spectroscopy results showed the preservation of mineral crystallinity and presence of residual organic material after all chemical treatments. A defatting step, which has not previously been used in conjunction with deproteinization to isolate the mineral phase, was also used. Finally, Raman spectroscopy demonstrated that the inclusion of a defatting procedure resulted in the removal of some but not all residual protein in the bone.