Optic Nerve Head Myelin-Related Protein, GFAP, and Iba1 Alterations in Non-Human Primates With Early to Moderate Experimental Glaucoma.

Purpose: The purpose of this study was to test if optic nerve head (ONH) myelin basic protein (MBP), 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), glial fibrillary acidic protein (GFAP), and ionized calcium binding adaptor molecule 1 (Iba1) proteins are altered in non-human primate (NHP) early/moderate experimental glaucoma (EG). Methods: Following paraformaldehyde perfusion, control and EG eye ONH tissues from four NHPs were paraffin embedded and serially (5 µm) vertically sectioned. Anti-MBP, CNPase, GFAP, Iba1, and nuclear dye-stained sections were imaged using sub-saturating light intensities. Whole-section images were segmented creating anatomically consistent laminar (L) and retrolaminar (RL) regions/sub-regions. EG versus control eye intensity/pixel-cluster density data within L and two RL regions (RL1 [1-250 µm]/RL2 [251-500 µm] from L) were compared using random effects models within the statistical program "R." Results: EG eye retinal nerve fiber loss ranged from 0% to 20%. EG eyes' MBP and CNPase intensity were decreased within the RL1 (MBP = 31.4%, P < 0.001; CNPase =62.3%, P < 0.001) and RL2 (MBP = 19.6%, P < 0.001; CNPase = 56.1%, P = 0.0004) regions. EG eye GFAP intensity was decreased in the L (41.6%, P < 0.001) and RL regions (26.7% for RL1, and 28.4% for RL2, both P < 0.001). Iba1+ and NucBlue pixel-cluster density were increased in the laminar (28.2%, P = 0.03 and 16.6%, P = 0.008) and both RL regions (RL1 = 37.3%, P = 0.01 and 23.7%, P = 0.0002; RL2 = 53.7%, P = 0.002 and 33.2%, P < 0.001). Conclusions: Retrolaminar myelin disruption occurs early in NHP EG and may be accompanied by laminar and retrolaminar decreases in astrocyte process labeling and increases in microglial/ macrophage density. The mechanistic and therapeutic implications of these findings warrant further study.

3D Histomorphometric Reconstruction and Quantification of the Optic Nerve Head Connective Tissues.

Accurately characterizing the 3D geometry of the optic nerve head neural and connective tissues has been the goal of a large and important body of scientific work. In the present report, we summarize our methods for the high-resolution, digital, 3D histomorphometric reconstruction of the optic nerve head tissues, including their visualization, parameterization, and quantification. In addition, we present our methods for between-eye comparisons of this anatomy, and their use to determine animal-specific and experiment-wide experimental glaucoma versus Control eye differences in the unilateral, monkey experimental glaucoma model. Finally, we demonstrate its application to finite element modeling, 3D optic nerve head reconstruction of other species, and 3D optic nerve head reconstructions using other imaging modalities.

The connective tissue phenotype of glaucomatous cupping in the monkey eye - Clinical and research implications.

In a series of previous publications we have proposed a framework for conceptualizing the optic nerve head (ONH) as a biomechanical structure. That framework proposes important roles for intraocular pressure (IOP), IOP-related stress and strain, cerebrospinal fluid pressure (CSFp), systemic and ocular determinants of blood flow, inflammation, auto-immunity, genetics, and other non-IOP related risk factors in the physiology of ONH aging and the pathophysiology of glaucomatous damage to the ONH. The present report summarizes 20 years of technique development and study results pertinent to the characterization of ONH connective tissue deformation and remodeling in the unilateral monkey experimental glaucoma (EG) model. In it we propose that the defining pathophysiology of a glaucomatous optic neuropathy involves deformation, remodeling, and mechanical failure of the ONH connective tissues. We view this as an active process, driven by astrocyte, microglial, fibroblast and oligodendrocyte mechanobiology. These cells, and the connective tissue phenomena they propagate, have primary and secondary effects on retinal ganglion cell (RGC) axon, laminar beam and retrolaminar capillary homeostasis that may initially be "protective" but eventually lead to RGC axonal injury, repair and/or cell death. The primary goal of this report is to summarize our 3D histomorphometric and optical coherence tomography (OCT)-based evidence for the early onset and progression of ONH connective tissue deformation and remodeling in monkey EG. A second goal is to explain the importance of including ONH connective tissue processes in characterizing the phenotype of a glaucomatous optic neuropathy in all species. A third goal is to summarize our current efforts to move from ONH morphology to the cell biology of connective tissue remodeling and axonal insult early in the disease. A final goal is to facilitate the translation of our findings and ideas into neuroprotective interventions that target these ONH phenomena for therapeutic effect.

Lamina Cribrosa Microarchitecture in Monkey Early Experimental Glaucoma: Global Change.

PURPOSE: The purpose of this study was to characterize experimental glaucoma (EG) versus control eye differences in lamina cribrosa (LC), beam diameter (BD), pore diameter (PD), connective tissue volume fraction (CTVF), connective tissue volume (CTV), and LC volume (LV) in monkey early EG. METHODS: Optic nerve heads (ONHs) of 14 unilateral EG and 6 bilateral normal (BN) monkeys underwent three-dimensional reconstruction and LC beam segmentation. Each beam and pore voxel was assigned a diameter based on the largest sphere that contained it before transformation to a common cylinder with inner, middle, and outer layers. Full-thickness and layer averages for BD, PD, CTVF, CTV, and LV were calculated for each ONH. Beam diameter and PD distributions for each ONH were fit to a gamma distribution and summarized by scale and shape parameters. Experimental glaucoma and depth effects were assessed for each parameter by linear mixed-effects (LME) modeling. Animal-specific EG versus control eye differences that exceeded the maximum intereye difference among the six BN animals were considered significant. RESULTS: Overall EG eye mean PD was 12.8% larger (28.2 ± 5.6 vs. 25.0 ± 3.3 µm), CTV was 26.5% larger (100.06 ± 47.98 vs. 79.12 ± 28.35 × 106 µm3), and LV was 40% larger (229.29 ± 98.19 vs. 163.63 ± 39.87 × 106 µm3) than control eyes (P ≤ 0.05, LME). Experimental glaucoma effects were significantly different by layer for PD (P = 0.0097) and CTVF (P < 0.0001). Pore diameter expanded consistently across all PDs. Experimental glaucoma eye-specific parameter change was variable in magnitude and direction. CONCLUSIONS: Pore diameter, CTV, and LV increase in monkey early EG; however, EG eye-specific change is variable and includes both increases and decreases in BD and CTVF.

The Connective Tissue Components of Optic Nerve Head Cupping in Monkey Experimental Glaucoma Part 1: Global Change.

PURPOSE: To characterize optic nerve head (ONH) connective tissue change within 21 monkey experimental glaucoma (EG) eyes, so as to identify its principal components. METHODS: Animals were imaged three to five times at baseline then every 2 weeks following chronic unilateral IOP elevation, and euthanized early through end-stage confocal scanning laser tomographic change. Optic nerve heads were serial-sectioned, three-dimensionally (3D) reconstructed, delineated, and quantified. Overall EG versus control eye differences were assessed by general estimating equations (GEE). Significant, animal-specific, EG eye change was required to exceed the maximum physiologic intereye differences in six healthy animals. RESULTS: Overall EG eye change was significant (P < 0.0026) and animal-specific EG eye change most frequent, for five phenomena (number of EG eyes and range of animal-specific change): posterior laminar deformation (21, -29 to -437 µm), laminar thickening (11, 20-73 µm) and thinning (3, -23 to -31 µm), scleral canal expansion (17, 20-139 µm), outward anterior (16, -16 to -124 µm) and posterior (17, -22 to -279 µm) laminar insertion migration, and peripapillary scleral bowing (11, 21-77 µm). Experimental glaucoma versus control eye laminar thickness differences were bimodal in behavior, being thickened in most EG eyes demonstrating the least deformation and less thickened or thinned in most EG eyes demonstrating the greatest deformation. CONCLUSIONS: Our postmortem studies retrospectively identify five connective tissue components of ONH "cupping" in monkey EG which serve as targets for longitudinally staging and phenotyping ONH connective tissue alteration within all forms of monkey and human optic neuropathy.

Age-related differences in longitudinal structural change by spectral-domain optical coherence tomography in early experimental glaucoma.

PURPOSE: To characterize age-related differences in the magnitude of spectral-domain optical coherence tomography (SD-OCT) structural change in early experimental glaucoma (EG). METHODS: Both eyes from four young (1.4-2.6 years) and four old (18.6-21.9 years) rhesus monkeys were imaged at least three times at baseline, and then every 2 weeks after laser-induced, chronic, unilateral IOP elevation until the onset of EG (confocal scanning laser tomographic surface change confirmed twice). Two to 20 weeks after EG onset, animals were euthanized and optic nerve axon counts for all eyes were performed. Masked operators delineated retinal and ONH landmarks in 40 radial B-scans from each eye and imaging session to quantify change from baseline in five SD-OCT neural and connective tissue parameters. The effects of EG, age, and EG × age interactions on the magnitude, rate (magnitude per postlaser time), and IOP responsiveness (magnitude per cumulative IOP insult) of postlaser parameter change were individually assessed using general estimating equation models. RESULTS: Presac SD-OCT RNFLT and minimum rim width change and postmortem axon loss was not significantly different in old compared with young EG eyes. The rate of change and IOP responsiveness of the parameters anterior lamina cribrosa surface depth relative to Bruch's membrane opening (BMO) and BMO depth relative to peripheral Bruch's membrane were significantly lower (P < 0.05) in the old compared with the young EG eyes. CONCLUSIONS: At similar postlaser times, levels of cumulative IOP insult and axonal damage, SD-OCT-detected ONH connective tissue structural change is greater in young compared with old monkey EG eyes.

Longitudinal detection of optic nerve head changes by spectral domain optical coherence tomography in early experimental glaucoma.

PURPOSE: We determined if the detection of spectral-domain optical coherence tomography (SDOCT) optic nerve head (ONH) change precedes the detection of confocal scanning laser tomography (CSLT) ONH surface, SDOCT retinal nerve fiber layer (RNFL), scanning laser perimetry (SLP), and multifocal electroretinography (mfERG) change in eight experimental glaucoma (EG) eyes. METHODS: Both eyes from eight monkeys were tested at least three times at baseline, and then every 2 weeks following laser-induced chronic unilateral IOP elevation. Event and trend-based definitions of onset in the control and EG eyes for 11 SDOCT neural and connective tissue, CSLT surface, SDOCT RNFL, SLP, and mfERG parameters were explored. The frequency and timing of onset for each parameter were compared using a logrank test. RESULTS: Maximum post-laser IOP was 18 to 42 mm Hg in the EG eyes and 12 to 20 mm Hg in the control eyes. For event- and trend-based analyses, onsets were achieved earliest and most frequently within the ONH neural and connective tissues using SDOCT, and at the ONH surface using CSLT. SDOCT ONH neural and connective tissue parameter change preceded or coincided with CSLT ONH surface change in most EG eyes. The SDOCT and SLP measures of RNFL thickness, and mfERG measures of visual function demonstrated similar onset rates, but occurred later than SDOCT ONH and CSLT surface change, and in fewer eyes. CONCLUSIONS: SDOCT ONH change detection commonly precedes or coincides with CSLT ONH surface change detection, and consistently precedes RNFLT, SLP, and mfERG change detection in monkey experimental glaucoma.

Posterior (outward) migration of the lamina cribrosa and early cupping in monkey experimental glaucoma.

PURPOSE: To quantify the lamina cribrosa insertion into the peripapillary sclera and optic nerve pia in normal (N) and early experimental glaucoma (EEG) monkey eyes. METHODS: Perfusion-fixed optic nerve heads (ONHs) from 21 animals were digitally reconstructed three dimensionally and delineated. Anterior Laminar Insertion Position (ALIP), Posterior Laminar Insertion Position (PLIP), Laminar Insertion Length (LIL; distance between the anterior and posterior laminar insertions), and Scleral Thickness (at the Anterior Sub-arachnoid space) were calculated for each ONH. Animals were pooled into four groups based on the kill condition (N vs. EEG) and perfusion IOP (10, 30, or 45 mm Hg) of each eye: N10-N10 (n = 6), N30/45-N10 (n = 6), EEG10-N10 (n = 3), and EEG30/45-N10 (n = 6). Glaucomatous EEG versus N eye differences in each group and each animal were required not only to achieve statistical significance (P < 0.05) but also to exceed physiologic intereye differences within the bilaterally normal groups. RESULTS: ALIP was significantly posterior (outward) in the EEG compared with N10 eyes of the EEG30/45-N10 group and 5 of 9 individual EEG eyes (difference range, 12-49 µm). PLIP was significantly posterior in the EEG eyes of both EEG groups and in 6 of 9 individual EEG eyes (range, 25-83 µm). LIL ranged from 90 to 190 µm in normal eyes and was significantly increased within the EEG eyes of both EEG groups and in 7 of 9 individual EEG eyes (difference range, 30-47 µm). CONCLUSIONS: Posterior migration of the lamina cribrosa is a component of early cupping in monkey EEG.

A comparison of optic nerve head morphology viewed by spectral domain optical coherence tomography and by serial histology.

PURPOSE: To compare serial optic nerve head (ONH) histology with interpolated B-scans generated from a three-dimensional (3-D) spectral domain (SD)-OCT ONH volume acquired in vivo from the same normal monkey eye. METHODS: A 15 degrees ONH SD-OCT volume was acquired in a normal monkey eye, with IOP manometrically controlled at 10 mm Hg. After perfusion fixation at 10 mm Hg, the ONH was trephined, the specimen embedded in a paraffin block, and serial sagittal sections cut at 4-mum intervals. The location of each histologic section was identified within the optic disc photograph by matching the position of the retinal vessels and of Bruch's membrane opening. By altering the angles of rotation and incidence, interpolated B-scans matching the location of the histologic sections were generated with custom software. Structures identified in the histologic sections were compared with signals identified in the matched B-scans. RESULTS: Close matches between histologic sections and interpolated B-scans were identified throughout the extent of the ONH. SD-OCT identified the neural canal opening as the termination of the Bruch's membrane-retinal pigment complex and border tissue as the innermost termination of the choroidal signal. The anterior lamina cribrosa and its continuity with the prelaminar glial columns were also detected by SD-OCT. CONCLUSIONS: Volumetric SD-OCT imaging of the ONH generates interpolated B-scans that accurately match serial histologic sections. SD-OCT captures the anterior laminar surface, which is likely to be a key structure in the detection of early ONH damage in ocular hypertension and glaucoma.

A novel type of sequence variation: multiple-nucleotide length polymorphisms discovered in the bovine genome.

Three types of sequence variations--single-nucleotide polymorphisms (SNPs), insertions and deletions (indels), and short tandem repeats (STRs)--have been extensively reported in mammalian genomes. In this study, we discovered a novel type of sequence variation, i.e., multiple-nucleotide length polymorphisms (MNLPs) in bovine UCN3 (Urocortin 3) and its receptor CRHR2 (corticotropin-releasing hormone receptor 2) genes. Both MNLPs featured involvement of multiple-nucleotide length polymorphisms (5-18 bases), low sequence identity, and 1.7- to 11-fold changes in promoter activity between two alleles. Therefore, this novel genetic complexity would contribute significantly to the evolutionary, functional, and phenotypic complexity of genomes within or among species.

Cross species association examination of UCN3 and CRHR2 as potential pharmacological targets for antiobesity drugs.

BACKGROUND: Obesity now constitutes a leading global public health problem. Studies have shown that insulin resistance affiliated with obesity is associated with intramyocellular lipid (IMCL) accumulation. Therefore, identification of genes associated with the phenotype would provide a clear target for pharmaceutical intervention and care for the condition. We hypothesized that urocortin 3 (UCN3) and corticotropin-releasing hormone receptor 2 (CRHR2) are associated with IMCL and subcutaneous fat depth (SFD), because the corticotropin-releasing hormone family of peptides are capable of strong anorectic and thermogenic effects. METHODOLOGY/PRINCIPAL FINDINGS: We annotated both bovine UCN3 and CRHR2 genes and identified 12 genetic mutations in the former gene and 5 genetic markers in the promoter region of the latter gene. Genotyping of these 17 markers on Wagyu times Limousin F(2) progeny revealed significant associations between promoter polymorphisms and SFD (P = 0.0203-0.0685) and between missense mutations of exon 2 and IMCL (P = 0.0055-0.0369) in the bovine UCN3 gene. The SFD associated promoter SNPs caused a gain/loss of 12 potential transcription regulatory binding sites, while the IMCL associated coding SNPs affected the secondary structure of UCN3 mRNA. However, none of five polymorphisms in CRHR2 gene clearly co-segregated with either trait in the population (P>0.6000). CONCLUSIONS/SIGNIFICANCE: Because UCN3 is located on human chromosome 10p15.1 where quantitative trait loci for obesity have been reported, our cross species study provides further evidence that it could be proposed as a potential target for developing antiobesity drugs. None of the markers in CRHR2 was associated with obesity-type traits in cattle, which is consistent with findings in human. Therefore, CRHR2 does not lend itself to the development of antiobesity drugs.