Comparison of the Biomechanics of the Mouse Astrocytic Lamina Cribrosa Between Glaucoma and Optic Nerve Crush Models.

Purpose: The strain response of the mouse astrocytic lamina (AL) to an ex vivo mechanical test was compared between two protocols: eyes that underwent sustained intraocular pressure (IOP) increase and eyes after optic nerve crush. Methods: Chronic IOP elevation was induced by microbead injection or the optic nerve was crushed in mice with widespread green fluorescence. After 3 days or 6 weeks, eyes were inflation tested by a published method of two-photon fluorescence to image the AL. Digital volume correlation was used to calculate strains. Optic nerve axon damage was also evaluated. Results: In the central AL but not the peripheral AL, four strains were greater in eyes at the 3-day glaucoma time point than control (P from 0.029 to 0.049, n = 8 eyes per group). Also, at this time point, five strains were greater in the central AL compared to the peripheral AL (P from 0.041 to 0.00003). At the 6-week glaucoma time point, the strains averaged across the specimen, in the central AL, and the peripheral AL were indistinguishable from the respective controls. Strains were not significantly different between controls and eyes 3 days or 6 weeks after crush (n = 8 and 16). Conclusions: We found alterations in the ex vivo mechanical behavior in eyes from mice with experimental glaucoma but not in those with crushed optic nerves. The results of this study demonstrate that significant axon injury does not directly affect mechanical behavior of the astrocytic lamina.

Association of clinical signs of chorioamnionitis with histological chorioamnionitis and neonatal outcomes.

BACKGROUND: Chorioamnionitis is a risk factor for fetal and neonatal outcomes. Therefore, predicting histological chorioamnionitis (HCA) and neonatal outcomes using clinical parameters could be helpful in management and preventing morbidities. OBJECTIVE: To determine if parameters of clinical chorioamnionitis (CCA) would be associated with HCA and neonatal outcomes. STUDY DESIGN: In this cohort study using a retrospective design, we analyzed the performance of signs of CCA in predicting HCA, and neonatal outcomes. Data were extracted from the electronic health record for all neonates with documented CCA delivered at our institution from 2011 to 2016. We compared our findings based on the old ACOG definition of CCA and the new definition released in 2017 - maternal fever plus any of fetal tachycardia, maternal leukocytosis, and purulent vaginal discharge. Maternal tachycardia and uterine tenderness were removed from the new criteria. Neonatal laboratory samples on admission, 12 h and 24 h were used to define the three time points of neonatal suspected sepsis. RESULTS: There were 530 mothers-infant dyads with chorioamnionitis. Seventy-three were preterm, and 457 were term. Eighty-eight percent of the preterm mothers had CCA, and HCA was present in 62.5% of 72 preterm placentas. Preterm infants with placental HCA significantly had lower birth weight, gestational age, placental weight, and more infants with lower 5-minute Apgar scores, compared to those with no HCA. In preterm infants, maternal urinary tract infection was significantly associated with decreased odds for HCA (OR 0.22, CI 0.10 - 0.71). More preterm babies with suspected sepsis criteria at the 3 time points had HCA (all p ≤ .01). In the term cohort, 95.4% and 65.6% had CCA and HCA, respectively. In term infants (n = 457), maternal leukocytosis (p = .002) and prolonged rupture of membranes (PROM; p = 002) were associated with HCA. Suspected sepsis was associated with PROM (p = .04), HCA (p = .0001), and maternal leukocytosis (p ≤ .05) in at least 1 of the 3 time points. CONCLUSION: Though maternal leukocytosis was significantly associated with the presence of HCA in the term cohort, there were no CCA criteria that accurately predicted presence of HCA in either the preterm or the term infants.

Aquaporin 4 is not present in normal porcine and human lamina cribrosa.

Aquaporin 4 is absent from astrocytes in the rodent optic nerve head, despite high expression in the retina and myelinated optic nerve. The purpose of this study was to quantify regional aquaporin channel expression in astrocytes of the porcine and human mouse optic nerve (ON). Ocular tissue sections were immunolabeled for aquaporins 1(AQP1), 4(AQP4), and 9(AQP9), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP) and alpha-dystroglycan (αDG) for their presence in retina, lamina, myelin transition zone (MTZ, region just posterior to lamina) and myelinated ON (MON). Semi- quantification of AQP4 labeling & real-time quantitative PCR (qPCR) data were analyzed in retina and ON tissue. Porcine and control human eyes had abundant AQP4 in Müller cells, retinal astrocytes, and myelinated ON (MON), but minimal expression in the lamina cribrosa. AQP1 and AQP9 were present in retina, but not in the lamina. Immunolabeling of GFAP and αDG was similar in lamina, myelin transition zone (MTZ) and MON regions. Semi-quantitative AQP4 labeling was at background level in lamina, increasing in the MTZ, and highest in the MON (lamina vs MTZ, MON; p≤0.05, p≤0.01, respectively). Expression of AQP4 mRNA was minimal in lamina and substantial in MTZ and MON, while GFAP mRNA expression was uniform among the lamina, MTZ, and MON regions. Western blot assay showed AQP4 protein expression in the MON samples, but none was detected in the lamina tissue. The minimal presence of AQP4 in the lamina is a specific regional phenotype of astrocytes in the mammalian optic nerve head.

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 age on mitochondria, axonal transport, and axonal degeneration after chronic IOP elevation using a murine ocular explant model.

The purpose of this study was to compare younger and older mice after chronic intraocular pressure (IOP) elevation lasting up to 4 days with respect to mitochondrial density, structure, and movement, as well as axonal integrity, in an ex vivo explant model. We studied 2 transgenic mouse strains, both on a C57BL/6J background, one expressing yellow fluorescent protein (YFP) in selected axons and one expressing cyan fluorescent protein (CFP) in all mitochondria. Mice of 4 months or 14 months of age were exposed to chronic IOP by anterior chamber microbead injection for 14 h, 1, 3, or 4 days. The optic nerve head of globe--optic nerve explants were examined by laser scanning microscopy. Mitochondrial density, structure, and movement were quantified in the CFP explants, and axonal integrity was quantified in YFP explants. In control mice, there was a trend towards decreased mitochondrial density (# per mm2) with age when comparing younger to older, control mice, but this was not significant (1947 ±â€¯653 vs 1412 ±â€¯356; p = 0.19). Mitochondrial density decreased after IOP elevation, significantly, by 31%, in younger mice (p = 0.04) but trending towards a decrease, by 22%, in older mice (p = 0.82) compared to age matched controls. Mitochondrial mean size was not altered after chronic IOP elevation for 14 h or more (p ≥ 0.16). When assessing mitochondrial movement, in younger mice, 5% were mobile at any given time; 4% in the anterograde direction and 1% retrograde. In younger untreated tissue, only 75% of explants had moving mitochondria (mean = 15.8 moving/explant), while after glaucoma induction only 24% of explants had moving mitochondria (mean = 4.2 moving/explant; difference from control, p = 0.03). The distance mitochondria traveled in younger mice was unchanged after glaucoma exposure, but in older glaucoma explants the distance traveled was less than half of older controls (p < 0.0003). In younger mice, mitochondrial speed increased after 14 h of elevated IOP (p = 0.006); however, in older glaucoma explants, movement was actually slower than controls (p = 0.02). In RGC-YFP explants, axonal integrity declined significantly after 4 days of IOP elevation to a similar degree in both younger and older mice. Older mice underwent greater loss of mitochondrial movement with chronic IOP elevation than younger mice, but suffered similar short-term axonal fragmentation in C57BL/6J mice. These transgenic strains, studied in explants, permit observations of alterations in intracellular structure and organelle activity in experimental glaucoma.

Regional Retinal Ganglion Cell Axon Loss in a Murine Glaucoma Model.

Purpose: To determine if retinal ganglion cell (RGC) axon loss in experimental mouse glaucoma is uniform in the optic nerve. Methods: Experimental glaucoma was induced for 6 weeks with a microbead injection model in CD1 (n = 78) and C57BL/6 (B6, n = 68) mice. From epoxy-embedded sections of optic nerve 1 to 2 mm posterior to the globe, total nerve area and regional axon density (axons/1600 µm2) were measured in superior, inferior, nasal, and temporal zones. Results: Control eyes of CD1 mice have higher axon density and more total RGCs than control B6 mice eyes. There were no significant differences in control regional axon density in all mice or by strain (all P > 0.2, mixed model). Exposure to elevated IOP caused loss of RGC in both strains. In CD1 mice, axon density declined without significant loss of nerve area, while B6 mice had less density loss, but greater decrease in nerve area. Axon density loss in glaucoma eyes was not significantly greater in any region in either mouse strain (both P > 0.2, mixed model). In moderately damaged CD1 glaucoma eyes, and CD1 eyes with the greatest IOP elevation exposure, density loss differed by region (P = 0.05, P = 0.03, mixed model) with the greatest loss in the temporal and superior regions, while in severely injured B6 nerves superior loss was greater than inferior loss (P = 0.01, mixed model, Bonferroni corrected). Conclusions: There was selectively greater loss of superior and temporal optic nerve axons of RGCs in mouse glaucoma at certain stages of damage. Differences in nerve area change suggest non-RGC responses differ between mouse strains.

A mouse ocular explant model that enables the study of living optic nerve head events after acute and chronic intraocular pressure elevation: Focusing on retinal ganglion cell axons and mitochondria.

We developed an explant model of the mouse eye and optic nerve that facilitates the study of retinal ganglion cell axons and mitochondria in the living optic nerve head (ONH) in an ex vivo environment. Two transgenic mouse strains were used, one expressing yellow fluorescent protein in selected axons and a second strain expressing cyan fluorescent protein in all mitochondria. We viewed an explanted mouse eye and optic nerve by laser scanning microscopy at and behind the ONH, the site of glaucoma injury. Explants from previously untreated mice were studied with the intraocular pressure (IOP) set artificially at normal or elevated levels for several hours. Explants were also studied from eyes that had undergone chronic IOP elevation from 14 h to 6 weeks prior to ex vivo study. Image analysis in static images and video of individual mitochondria or axonal structure determined effects of acute and chronic IOP elevation. At normal IOP, fluorescent axonal structure was stable for up to 3 h under ex vivo conditions. After chronic IOP elevation, axonal integrity index values indicated fragmentation of axon structure in the ONH. In mice with fluorescent mitochondria, the normal density decreased with distance behind the ONH by 45% (p = 0.002, t-test). Density increased with prior chronic IOP elevation to 21,300 ± 4176 mitochondria/mm2 compared to control 16,110 ± 3159 mitochondria/mm2 (p = 0.025, t-test), but did not increase significantly after 4 h, acute IOP elevation (1.5% decrease in density, p = 0.83, t-test). Mean normal mitochondrial length of 2.3 ± 1.4 µm became 13% smaller after 4 h of IOP elevation ex vivo compared to baseline (p = 0.015, t-test, N-10). Normal mitochondrial speed of movement was significantly slower in the anterograde direction (towards the brain) than retrograde, but there were more mitochondria in motion and traveling longer lengths in anterograde direction. The percent of mitochondria in motion decreased by >50% with acute IOP increase to 30 mm Hg after 60 min. A new ocular explant model implemented with eyes from transgenic mice with fluorescent cellular components provided real time measurement of the early events in experimental glaucoma and quantitative outcomes for neuroprotection therapy experiments.

Measuring Deformation in the Mouse Optic Nerve Head and Peripapillary Sclera.

Purpose: To develop an ex vivo explant system using multiphoton microscopy and digital volume correlation to measure the full-field deformation response to intraocular pressure (IOP) change in the peripapillary sclera (PPS) and in the optic nerve head (ONH) astrocytic structure. Methods: Green fluorescent protein (GFP)-glutamate transporter-GLT1 (GLT1/GFP) mouse eyes were explanted and imaged with a laser-scanning microscope under controlled inflation. Images were analyzed for regional strains and changes in astrocytic lamina and PPS shape. Astrocyte volume fraction in seven control GLT1/GFP mice was measured. The level of fluorescence of GFP fluorescent astrocytes was compared with glial fibrillary acidic protein (GFAP) labeled astrocytes using immunohistochemistry. Results: The ONH astrocytic structure remained stable during 3 hours in explants. Control strain-globally, in the central one-half or two-thirds of the astrocytic lamina-was significantly greater in the nasal-temporal direction than in the inferior-superior or anterior-posterior directions (each P≤ 0.03, mixed models). The PPS opening (perimeter) in normal eye explants also became wider nasal-temporally than superior-inferiorly during inflation from 10 to 30 mm Hg (P = 0.0005). After 1 to 3 days of chronic IOP elevation, PPS area was larger than in control eyes (P = 0.035), perimeter elongation was 37% less than controls, and global nasal-temporal strain was significantly less than controls (P = 0.007). Astrocyte orientation was altered by chronic IOP elevation, with processes redirected toward the longitudinal axis of the optic nerve. Conclusions: The explant inflation test measures the strain response of the mouse ONH to applied IOP. Initial studies indicate regional differences in response to both acute and chronic IOP elevation within the ONH region.

The pressure-induced deformation response of the human lamina cribrosa: Analysis of regional variations.

The objective of this study was to measure the pressure-induced deformation response of the human lamina cribrosa (LC) and analyze for variations with age and anatomical region. The posterior scleral cup of 8 eyes from 6 human donors was mounted onto a custom inflation chamber. A laser-scanning microscope was used for second harmonic generation (SHG) imaging of the collagen structure in the posterior volume of the LC at pressures from 5mmHg to 45mmHg. The SHG volumes were analyzed by the Fast-Fourier Iterative Digital Volume Correlation (DVC) algorithm for the three dimensional (3D) displacement field. The components of the Green-Lagrange strain tensor and the in-plane principal and maximum shear strains were evaluated from the DVC displacement field for the central and peripheral regions of the LC and the nasal, temporal, inferior, and superior quadrants surrounding the central retinal artery and vein. Among the major findings were that older age was associated with lower strains, the maximum shear strain was larger in the peripheral than central region, and the maximum principal strain was lower in the nasal quadrant. The elliptical shape of the LC was also predictive of the biaxial strain ratio. Age-related and structure-related variations in the pressure-induced strains of the LC may contribute to the susceptibility and severity of optic nerve damage in glaucoma, and regional variations may explain the progression of axonal damage and tissue remodeling observed in the LC in glaucoma. STATEMENT OF SIGNIFICANCE: Glaucoma causes vision loss through progressive damage of the retinal ganglion axons at the lamina cribrosa (LC), the connective tissue structure that supports the axons as they leave the eye. Mechanical characterization of the LC is challenging because of the complex 3D shape and inaccessibility of the tissue. We present a new method using digital volume correlation to map the 3D displacement and strain fields in the LC under inflation. We report for the first time significant regional variations in the strains that are consistent with the pattern of optic nerve damage in early glaucoma. Thus regional strain variations may be predictive of the progression of axonal damage in glaucoma.

Scleral fibroblast response to experimental glaucoma in mice.

PURPOSE: To study the detailed cellular and molecular changes in the mouse sclera subjected to experimental glaucoma. METHODS: Three strains of mice underwent experimental bead-injection glaucoma and were euthanized at 3 days and 1, 3, and 6 weeks. Scleral protein expression was analyzed with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using (16)O/(18)O labeling for quantification in 1- and 6-week tissues. Sclera protein samples were also analyzed with immunoblotting with specific antibodies to selected proteins. The proportion of proliferating scleral fibroblasts was quantified with Ki67 and 4',6-diamidino-2-phenylindole (DAPI) labeling, and selected proteins were studied with immunohistochemistry. RESULTS: Proteomic analysis showed increases in molecules involved in integrin-linked kinase signaling and actin cytoskeleton signaling pathways at 1 and 6 weeks after experimental glaucoma. The peripapillary scleral region had more fibroblasts than equatorial sclera (p=0.001, n=217, multivariable regression models). There was a sixfold increase in proliferating fibroblasts in the experimental glaucoma sclera at 1 week and a threefold rise at 3 and 6 weeks (p=0.0005, univariate regression). Immunoblots confirmed increases for myosin, spectrin, and actinin at 1 week after glaucoma. Thrombospondin-1 (TSP-1), HINT1, vimentin, actinin, and α-smooth muscle actin were increased according to immunohistochemistry. CONCLUSIONS: Scleral fibroblasts in experimental mouse glaucoma show increases in actin cytoskeleton and integrin-related signaling, increases in cell division, and features compatible with myofibroblast transition.

Experimental scleral cross-linking increases glaucoma damage in a mouse model.

The purpose of this study was to assess the effect of a scleral cross-linking agent on susceptibility to glaucoma damage in a mouse model.CD1 mice underwent 3 subconjunctival injections of 0.5 M glyceraldehyde (GA) in 1 week, then had elevated intraocular pressure (IOP) induced by bead injection. Degree of cross-linking was measured by enzyme-linked immunosorbent assay (ELISA), scleral permeability was measured by fluorescence recovery after photobleaching (FRAP), and the mechanical effects of GA exposure were measured by inflation testing. Control mice had buffer injection or no injection in 2 separate glaucoma experiments. IOP was monitored by Tonolab and retinal ganglion cell (RGC) loss was measured by histological axon counting. To rule out undesirable effects of GA, we performed electroretinography and detailed histology of the retina. GA exposure had no detectable effects on RGC number, retinal structure or function either histologically or electrophysiologically. GA increased cross-linking of sclera by 37% in an ELISA assay, decreased scleral permeability (FRAP, p = 0.001), and produced a steeper pressure-strain behavior by in vitro inflation testing. In two experimental glaucoma experiments, GA-treated eyes had greater RGC axon loss from elevated IOP than either buffer-injected or control eyes, controlling for level of IOP exposure over time (p = 0.01, and 0.049, multivariable regression analyses). This is the first report that experimental alteration of the sclera, by cross-linking, increases susceptibility to RGC damage in mice.

Changes in scleral collagen organization in murine chronic experimental glaucoma.

PURPOSE: The organization of scleral collagen helps to determine the eye's biomechanical response to intraocular pressure (IOP), and may therefore be important in glaucoma. This study provides a quantitative assessment of changes in scleral collagen fibril organization in bead-induced murine experimental glaucoma. METHODS: Wide-angle X-ray scattering was used to study the effect of bead-induced glaucoma on posterior scleral collagen organization in one eye of 12 CD1 mice, with untreated fellow eyes serving as controls. Three collagen parameters were measured: the local preferred fibril directions, the degree of collagen anisotropy, and the total fibrillar collagen content. RESULTS: The mouse sclera featured a largely circumferential orientation of fibrillar collagen with respect to the optic nerve head canal. Localized alteration to fibril orientations was evident in the inferior peripapillary sclera of bead-treated eyes. Collagen anisotropy was significantly (P<0.05) reduced in bead-treated eyes in the superior peripapillary (Treated: 43±8%; CONTROL: 49±6%) and midposterior (Treated: 39±4%; CONTROL: 43±4%) sclera, and in the peripapillary region overall (Treated: 43±6%; CONTROL: 47±3%). No significant differences in total collagen content were found between groups. CONCLUSIONS: Spatial changes in collagen fibril anisotropy occur in the posterior sclera of mice with bead-induced chronic IOP elevation and axonal damage. These results support the idea that dynamic changes in scleral form and structure play a role in the development of experimental glaucoma in mice, and potentially in human glaucoma.

Scleral permeability varies by mouse strain and is decreased by chronic experimental glaucoma.

PURPOSE: To determine differences in scleral permeability, as measured by diffusion of macromolecules, by using fluorescence recovery after photobleaching (FRAP), with reference to differences by mouse strain, scleral region, and the effect of experimental glaucoma. METHODS: In three mouse strains (B6, CD1, and B6 mice with mutation in collagen 8α2 [Aca23]), we used FRAP to measure the diffusion of fluorescein isothiocyanate-dextran, molecular weight 40 kDa, into a photobleached zone of sclera. Scleral regions near the optic nerve head (peripapillary) and two successively more anterior regions were compared. Sclera from mouse eyes subjected to chronically elevated intraocular pressure after bead injection into the anterior chamber were compared to fellow eye controls. FRAP data were compared against estimated retinal ganglion cell axon loss in glaucomatous eyes. RESULTS: Diffusion rates of dextran molecules in the sclera were significantly greater in Aca23 and B6 mice than in CD1 mice in a multivariate model adjusted for region and axial length (P < 0.0001). Dextran diffusion significantly decreased in glaucomatous eyes, and the decline increased with greater axon loss (P = 0.0003, multivariable model). Peripapillary scleral permeability was higher in CD1 than B6 and Aca23 mice (P < 0.05, multivariable model, adjusted by Bonferroni). CONCLUSIONS: Measurement of the diffusion rates of dextran molecules in the sclera showed that glaucoma leads to decreased scleral permeability in all three mouse strains tested. Among mouse strains tested, those that were more susceptible to glaucomatous loss of retinal ganglion cells had a lower scleral permeability at baseline.

Susceptibility to glaucoma damage related to age and connective tissue mutations in mice.

The purpose of this research was to study the effects of age and genetic alterations in key connective tissue proteins on susceptibility to experimental glaucoma in mice. We used mice haploinsufficient in the elastin gene (EH) and mice without both alleles of the fibromodulin gene (FM KO) and their wild type (WT) littermates of B6 and CD1 strains, respectively. FM KO mice were tested at two ages: 2 months and 12 months. Intraocular pressure (IOP) was measured by Tonolab tonometer, axial lengths and widths measured by digital caliper post-enucleation, and chronic glaucoma damage was measured using a bead injection model and optic nerve axon counts. IOP in EH mice was not significantly different from WT, but FM KO were slightly lower than their controls (p = 0.04). Loss of retinal ganglion cell (RGC) axons was somewhat, but not significantly greater in young EH and younger or older FM KO strains than in age-matched controls (p = 0.48, 0.34, 0.20, respectively, multivariable regression adjusting for IOP exposure). Older CD1 mice lost significantly more RGC axons than younger CD1 (p = 0.01, multivariable regression). The CD1 mouse strain showed age-dependence of experimental glaucoma damage to RGC in the opposite, and more expected, direction than in B6 mice in which older mice are more resistant to damage. Genetic alteration in two genes that are constituents of sclera, fibromodulin and elastin do not significantly affect RGC loss.

Scleral structural alterations associated with chronic experimental intraocular pressure elevation in mice.

PURPOSE: To study changes in scleral structure induced by chronic experimental intraocular pressure elevation in mice. METHODS: We studied the effect of chronic bead-induced glaucoma on scleral thickness, collagen lamellar structure, and collagen fibril diameter distribution in C57BL/6 (B6) and CD1 mice, and in collagen 8α2 mutant mice (Aca23) and their wild-type littermates (Aca23-WT) using electron and confocal microscopy. RESULTS: In unfixed tissue, the control B6 peripapillary sclera was thicker than in CD1 mice (p<0.001). After 6 weeks of glaucoma, the unfixed CD1 and B6 sclera thinned by 9% and 12%, respectively (p<0.001). The fixed sclera, measured by electron microscopy, was significantly thicker in control Aca23 than in B6 or CD1 mice (p<0.05). The difference between fresh and fixed scleral thickness was nearly 68% in untreated control B6 and CD1 mice, but differed by only 10% or less in fresh/fixed glaucoma scleral comparisons. There were 39.3±9.6 lamellae (mean, standard deviation) in control sclera, categorized as 41% cross-section, 24% cellular, 20% oblique, and 15% longitudinal. After glaucoma, mean peripapillary thickness significantly increased in fixed specimens of all mouse strains by 10.3 ±4.8 µm (p=0.001) and the total number of lamellae increased by 18% (p=0.01). The number of cellular and cross-section lamellae increased in glaucoma eyes. After glaucoma, there were more small and fewer large collagen fibrils (p<0.0001). Second harmonic generation imaging showed that the normal circumferential pattern of collagen fibrils in the peripapillary sclera was altered in significantly damaged glaucomatous eyes. CONCLUSIONS: Dynamic responses of the sclera to experimental mouse glaucoma may be more important than baseline anatomic features in explaining susceptibility to damage. These include decreases in nonfibrillar elements, alterations in lamellar orientation, an increased number of smaller collagen fibrils and fewer larger fibrils, and relative increase in the number of scleral fibroblast layers.

Studies of scleral biomechanical behavior related to susceptibility for retinal ganglion cell loss in experimental mouse glaucoma.

PURPOSE: To study anatomical changes and mechanical behavior of the sclera in mice with experimental glaucoma by comparing CD1 to B6 mice. METHODS: Chronic experimental glaucoma for 6 weeks was produced in 2- to 4-month-old CD1 (43 eyes) and B6 mice (42 eyes) using polystyrene bead injection into the anterior chamber with 126 control CD1 and 128 control B6 eyes. Intraocular pressure (IOP) measurements were made with the TonoLab at baseline and after bead injection. Axial length and scleral thickness were measured after sacrifice in the CD1 and B6 animals and compared to length data from 78 eyes of DBA/2J mice. Inflation testing of posterior sclera was conducted, and circumferential and meridional strain components were determined from the displacement response. RESULTS: Experimental glaucoma led to increases in axial length and width by comparison to fellow eyes (6% in CD1 and 10% in B6; all P < 0.03). While the peripapillary sclera became thinner in both mouse types with glaucoma, the remainder of the sclera uniformly thinned in CD1, but thickened in B6. Peripapillary sclera in CD1 controls had significantly greater temporal meridional strain than B6 and had differences in the ratios of meridional to effective circumferential strain from B6 mice. In both CD1 and B6 mice, exposure to chronic IOP elevation resulted in stiffer pressure-strain responses for both the effective circumferential and meridional strains (multivariable regression model, P = 0.01-0.03). CONCLUSIONS: Longer eyes, greater scleral strain in some directions at baseline, and generalized scleral thinning after glaucoma were characteristic of CD1 mice that have greater tendency to retinal ganglion cell damage than B6 mice. Increased scleral stiffness after glaucoma exposure in mice mimics findings in monkey and human glaucoma eyes.

Mice with an induced mutation in collagen 8A2 develop larger eyes and are resistant to retinal ganglion cell damage in an experimental glaucoma model.

PURPOSE: To study susceptibility to glaucoma injury as it may be affected by mutations in ocular connective tissue components. METHODS: Mice homozygous for an N-ethyl-N-nitrosourea induced G257D exchange (Gly to Asp) missense mutation (Aca23) in their collagen 8A2 gene were studied to measure intraocular pressure (IOP), axial length and width, number of retinal ganglion cells (RGC), and inflation responses. Three month old homozygous Aca23 mutant and wild type (WT) mice had 6 weeks exposure to elevated IOP induced by polystyrene microbead injection. Additional Aca23 and matched controls were studied at ages of 10 and 18 months. RESULTS: Aca23 mice had no significant difference from WT in IOP level, and in both strains IOP rose with age. In multivariable models, axial length and width were significantly larger in Aca23 than WT, became larger with age, and were larger after exposure to glaucoma (n=227 mice). From inflation test data, the estimates of scleral stress resultants in Aca23 mice were similar to age-matched and younger WT C57BL/6 (B6) mice, while the strain estimates for Aca23 were significantly less than those for either WT group in the mid-sclera and in some of the more anterior scleral measures (p<0.001; n=29, 22, 20 eyes in Aca23, older WT, younger WT, respectively). With chronic IOP elevation, Aca23 eyes increased 9% in length and 7% in width, compared to untreated fellow eyes (p<0.05, <0.01). With similar elevated IOP exposure, WT eyes enlarged proportionately twice as much as Aca23, increasing in length by 18% and in nasal-temporal width by 13% (both p<0.001, Mann-Whitney test). In 4 month old control optic nerves, mean RGC axon number was not different in Aca23 and WT (46,905±7,592, 43,628±11,162, respectively; p=0.43, Mann-Whitney test, n=37 and 29). With chronic glaucoma, Aca23 mice had a mean axon loss of only 0.57±17%, while WT mice lost 21±31% (median loss: 1% versus 10%, n=37, 29, respectively; p=0.001; multivariable model adjusting for positive integral IOP exposure). CONCLUSIONS: The Aca23 mutation in collagen 8α2 is the first gene defect found to alter susceptibility to experimental glaucoma, reducing RGC loss possibly due to differences in mechanical behavior of the sclera. Detailed study of the specific changes in scleral connective tissue composition and responses to chronic IOP elevation in this strain could produce new therapeutic targets for RGC neuroprotection.

Retinal ganglion cell morphology after optic nerve crush and experimental glaucoma.

PURPOSE: To study sequential changes in retinal ganglion cell (RGC) morphology in mice after optic nerve crush and after induction of experimental glaucoma. METHODS: Nerve crush or experimental glaucoma was induced in mice that selectively express yellow fluorescent protein (YFP) in RGCs. Mice were euthanized 1, 4, and 9 days after crush and 1, 3, and 6 weeks after induction of glaucoma by bead injection. All YFP-RGCs were identified in retinal whole mounts. Then confocal images of randomly selected RGCs were quantified for somal fluorescence brightness, soma size, neurite outgrowth, and dendritic complexity (Sholl analysis). RESULTS: By 9 days after crush, 98% of RGC axons died and YFP-RGCs decreased by 64%. After 6 weeks of glaucoma, 31% of axons died, but there was no loss of YFP-RGC bodies. All crush retinas combined had significant decreases in neurite outgrowth parameters (P ≤ 0.036, generalized estimating equation [GEE] model) and dendritic complexity was lower than controls (P = 0.017, GEE model). There was no change in RGC soma area after crush. In combined glaucoma data, the RGC soma area was larger than control (P = 0.04, GEE model). At 3 weeks, glaucoma RGCs had significantly larger values for dendritic structure and complexity than controls (P = 0.044, GEE model), but no statistical difference was found at 6 weeks. CONCLUSIONS: After nerve crush, RGCs and axons died rapidly, and dendritic structure decreased moderately in remaining RGCs. Glaucoma caused an increase in RGC dendrite structure and soma size at 3 weeks.

The effects of anesthesia, mouse strain and age on intraocular pressure and an improved murine model of experimental glaucoma.

The purpose of this study was to improve a mouse model of chronic intraocular pressure (IOP) elevation utilizing microbead injection in two strains of mice and to assess the effect of age and anesthesia on measured IOP. We compared our previous model with two modified protocols for injecting polystyrene microbeads and viscoelastic material in CD1or C57BL/6 mice. The measured outcomes were degree of IOP elevation and production of axonal loss. The first new protocol was injection of 3 µL of equal volumes of 6 µm and 1 µm diameter beads, followed by 2 µL of viscoelastic (3+2). The second new protocol injected 4 µL of the two bead mixture, then 1 µL of viscoelastic (4+1). Both were compared to injection of 2 µL of 6 µm beads with 3 µL of viscoelastic (2+3). We also compared the effects of age and of two anesthetic regimens (intraperitoneal ketamine/xylazine/acepromazine versus isoflurane gas) on measured IOP in untreated eyes of both strains. IOP was 2mm Hg lower with intraperitoneal than with gas anesthesia in both strains (p=0.003, p<0.0001, t-test). IOP measurements were lower in untreated young (2 months) compared to older (10 months) C57BL/6 mice (p=0.001, t-test). In the experimental glaucoma mouse model, mean IOP and number of elevated IOP measurements were higher in newer protocols. Mean axon loss with the 4+1 protocol (all strains) was twice that of the 2+3 and 3+2 protocols (36% vs. 15% loss, p=0.0026, ANOVA), and mean axon loss in CD1 mice (21%) was greater than in C57BL/6 mice (13%) (p=0.047, ANOVA). Median axon loss in 4+1 protocol treated C57BL/6 mice expressing yellow fluorescent protein in 2% of retinal ganglion cells (RGCs) had greater median axon loss than C57BL/6 4+1 protocol treated mice (26% vs. 10%, p=0.03). The 4+1 protocol provided higher, more consistent IOP elevation and greater axonal loss. The effects of age, strain, and anesthesia on induced IOP elevation and axon damage must be considered in mouse experimental glaucoma research.

Semi-automated, quantitative analysis of retinal ganglion cell morphology in mice selectively expressing yellow fluorescent protein.

The development of transgenic mouse lines that selectively label a subset of neurons provides unique opportunities to study detailed neuronal morphology and morphological changes under experimental conditions. In the present study, a mouse line in which a small number of retinal ganglion cells (RGCs) express yellow fluorescent protein (YFP) under control of the Thy-1 promoter was used (Feng et al., 2000). We characterized the number, distribution by retinal region and eccentricity of YFP-labeled RGCs using fluorescence microscopy and Stereo Investigator software (MicroBrightField, VT, USA). Then, we captured images of 4-6 YFP-expressing RGCs from each of 8 retinal regions by confocal microscopy, producing 3-dimensional and flattened data sets. A new semi-automated method to quantify the soma size, dendritic length and dendritic arbor complexity was developed using MetaMorph software (Molecular Devices, PA, USA). Our results show that YFP is expressed in 0.2% of all RGCs. Expression of YFP was not significantly different in central versus peripheral retina, but there were higher number of YFP-expressing RGCs in the temporal quadrant than in the nasal. By confocal-based analysis, 58% of RGCs expressing YFP did so at a high level, with the remainder distributed in decreasing levels of brightness. Variability in detailed morphometric parameters was as great between two fellow retinas as in retinas from different mice. The analytic methods developed for this selective YFP-expressing RGC model permit quantitative comparisons of parameters relevant to neuronal injury.