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.

Characterizing the collagen network structure and pressure-induced strains of the human lamina cribrosa. Invest

The effects of glaucoma on the pressure-induced strain response of the human lamina cribrosa. Invest

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 HO, 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.

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.