Stretch-Induced Uncrimping of Equatorial Sclera Collagen Bundles.

Stretch-induced collagen uncrimping underlies the nonlinear mechanical behavior of the sclera according to what is often called the process of recruitment. We recently reported experimental measurements of sclera collagen crimp and pressure-induced uncrimping. Our studies, however, were cross-sectional, providing statistical descriptions of crimp with no information on the effects of stretch on specific collagen bundles. Data on bundle-specific uncrimping is necessary to better understand the effects of macroscale input on the collagen microscale and tissue failure. Our goal in this project was to measure bundle-specific stretch-induced collagen uncrimping of sclera. Three goat eyes were cryosectioned sagittally (30 µm). Samples of equatorial sclera were isolated, mounted to a custom uni-axial stretcher and imaged with polarized light microscopy at various levels of clamp-to-clamp stretch until failure. At each stretch level, local strain was measured using image tracking techniques. The level of collagen crimping was determined from the bundle waviness, defined as the circular standard deviation of fiber orientation along a bundle. Eye-specific recruitment curves were then computed using eye-specific waviness at maximum stretch before sample failure to define fibers as recruited. Nonlinear mixed effect models were used to determine the associations of waviness to local strain and recruitment to clamp-to-clamp stretch. Waviness decreased exponentially with local strain (p < 0.001), whereas bundle recruitment followed a sigmoidal curve with clamp-to-clamp stretch (p < 0.001). Individual bundle responses to stretch varied substantially, but recruitment curves were similar across sections and eyes. In conclusion, uni-axial stretch caused measurable bundle-specific uncrimping, with the sigmoidal recruitment pattern characteristic of fiber-reinforced soft tissues.

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.

Crimp around the globe; patterns of collagen crimp across the corneoscleral shell.

Our goal was to systematically quantify the collagen crimp morphology around the corneoscleral shell, and test the hypothesis that collagen crimp is not uniform over the globe. Axial longitudinal cryosections (30 µm) of three sheep eyes, fixed at 0 mmHg IOP, were imaged using polarized light microscopy to quantify the local collagen in 8 regions: two corneal (central and peripheral) and six scleral (limbus, anterior-equatorial, equatorial, posterior-equatorial, posterior and peripapillary). Collagen crimp period (length of one wave), tortuosity (path length divided by end-to-end length), waviness (SD of orientation), amplitude (half the peak to trough distance), and conformity (width of contiguous similarly oriented bundles) were measured in each region. Measurements were obtained on 8216 collagen fiber bundles. When pooling measurements across the whole eye globe, the median crimp values were: 23.9 µm period, 13.2 µm conformity, 0.63 µm amplitude, 1.006 tortuosity, and 12.7° waviness. However, all parameters varied significantly across the globe. Median bundle periods in the central cornea, limbus, and peripapillary sclera (PPS) were 14.1 µm, 29.5 µm, and 22.9 µm, respectively. Median conformities were 20.8 µm, 14.5 µm, and 15.1 µm, respectively. Median tortuosities were 1.005, 1.007, and 1.007, respectively. Median waviness' were 11.4°, 13.2°, and 13.2°, respectively. Median amplitudes were 0.35 µm, 0.87 µm, and 0.65 µm, respectively. All parameters varied significantly across the globe. All regions differed significantly from one another on at least one parameter. Regions with small periods had large conformities, and bundles with high tortuosity had high waviness and amplitude. Waviness, tortuosity, and amplitude, associated with nonlinear biomechanical behavior, exhibited "double hump" distributions, whereas period and conformity, representing tissue organization, were substantially different between sclera and cornea. Though the biomechanical implications and origin of the patterns observed remain unclear, our findings of well-defined patterns of collagen crimp across the corneoscleral shell, consistent between eyes, support the existence of mechanisms that regulate collagen characteristics at the regional or smaller levels. These results are experimental data necessary for more realistic models of ocular biomechanics and remodeling.

CenTauR: Toward a universal scale and masks for standardizing tau imaging studies.

INTRODUCTION: Recently, an increasing number of tau tracers have become available. There is a need to standardize quantitative tau measures across tracers, supporting a universal scale. We developed several cortical tau masks and applied them to generate a tau imaging universal scale. METHOD: One thousand forty-five participants underwent tau scans with either 18F-flortaucipir, 18F-MK6240, 18F-PI2620, 18F-PM-PBB3, 18F-GTP1, or 18F-RO948. The universal mask was generated from cognitively unimpaired amyloid beta (Aß)- subjects and Alzheimer's disease (AD) patients with Aß+. Four additional regional cortical masks were defined within the constraints of the universal mask. A universal scale, the CenTauRz, was constructed. RESULTS: None of the regions known to display off-target signal were included in the masks. The CenTauRz allows robust discrimination between low and high levels of tau deposits. DISCUSSION: We constructed several tau-specific cortical masks for the AD continuum and a universal standard scale designed to capture the location and degree of abnormality that can be applied across tracers and across centers. The masks are freely available at https://www.gaain.org/centaur-project.

Biostatistical Estimation of Tau Threshold Hallmarks (BETTH) Algorithm for Human Tau PET Imaging Studies.

A methodology for determining tau PET thresholds is needed to confidently detect early tau deposition. We compared multiple threshold-determining methods in participants who underwent either 18F-flortaucipir or 18F-MK-6240 PET scans. Methods: 18F-flortaucipir (n = 798) and 18F-MK-6240 (n = 216) scans were processed and sampled to obtain regional SUV ratios. Subsamples of the cohorts were based on participant diagnosis, age, amyloid-ß status (positive or negative), and neurodegeneration status (positive or negative), creating older-adult (age ≥ 55 y) cognitively unimpaired (amyloid-ß-negative, neurodegeneration-negative) and cognitively impaired (mild cognitive impairment/Alzheimer disease, amyloid-ß-positive, neurodegeneration-positive) groups, and then were further subsampled via matching to reduce significant differences in diagnostic prevalence, age, and Mini-Mental State Examination score. We used the biostatistical estimation of tau threshold hallmarks (BETTH) algorithm to determine sensitivity and specificity in 6 composite regions. Results: Parametric double receiver operating characteristic analysis yielded the greatest joint sensitivity in 5 of the 6 regions, whereas hierarchic clustering, gaussian mixture modeling, and k-means clustering all yielded perfect joint specificity (2.00) in all regions. Conclusion: When 18F-flortaucipir and 18F-MK-6240 are used, Alzheimer disease-related tau status is best assessed using 2 thresholds, a sensitivity one based on parametric double receiver operating characteristic analysis and a specificity one based on gaussian mixture modeling, delimiting an uncertainty zone indicating participants who may require further evaluation.

Direct Comparison of the Tau PET Tracers 18F-Flortaucipir and 18F-MK-6240 in Human Subjects.

Tau PET tracers exhibit varying levels of specific signal and distinct off-target binding patterns that are more diverse than amyloid PET tracers. This study compared 2 frequently used tau PET tracers, 18F-flortaucipir and 18F-MK-6240, in the same subjects. Methods:18F-flortaucipir and 18F-MK-6240 scans were collected within 2 mo in 15 elderly subjects varying in clinical diagnosis and cognition. FreeSurfer, version 5.3, was applied to 3-T MR images to segment Braak pathologic regions (I-VI) for PET analyses. Off-target binding was assessed in the choroid plexus, meninges, and striatum. SUV ratio (SUVR) outcomes were determined over 80-100 min (18F-flortaucipir) or 70-90 min (18F-MK-6240) normalized to cerebellar gray matter. Masked visual interpretation of images was performed by 5 raters for both the medial temporal lobe and the neocortex, and an overall (majority) rating was determined. Results: Overall visual ratings showed complete concordance between radiotracers for both the medial temporal lobe and the neocortex. SUVR outcomes were highly correlated (r2 > 0.92; P ≪ 0.001) for all Braak regions except Braak II. The dynamic range of SUVRs in target regions was approximately 2-fold higher for 18F-MK-6240 than for 18F-flortaucipir. Cerebellar SUVs were similar for 18F-MK-6240 and 18F-flortaucipir, suggesting that differences in SUVRs are driven by specific signals. Apparent off-target binding was observed often in the striatum and choroid plexus with 18F-flortaucipir and most often in the meninges with 18F-MK-6240. Conclusion: Both 18F-MK-6240 and 18F-flortaucipir are capable of quantifying signal in a common set of brain regions that develop tau pathology in Alzheimer disease; these tracers perform equally well in visual interpretations. Each also shows distinct patterns of apparent off-target binding. 18F-MK-6240 showed a greater dynamic range in SUVR estimates, which may be an advantage in detecting early tau pathology or in performing longitudinal studies to detect small interval changes.

Radial and Circumferential Collagen Fibers Are a Feature of the Peripapillary Sclera of Human, Monkey, Pig, Cow, Goat, and Sheep.

Purpose: To test the hypothesis that human, monkey, pig, sheep, cow, and goat eyes exhibit circumferential, radial, and interweaving collagen architecture in the posterior sclera. Methods: We analyzed 1,327 cryosections from the posterior poles of 4 human, 4 monkey, 5 pig, 8 sheep, 1 goat, and 2 cow eyes. Images were acquired using polarized light microscopy and processed to obtain polar fiber orientations relative to the center of the canal. Circumferential, radial, and interweaving regions were identified and analyzed for mean fiber orientation and anisotropy and region width and thickness. Results: Every eye exhibited circumferential, radial, and interweaving fibers in consistent locations. Radial fibers extended out from near the canal into the peripapillary and peripheral sclera in the innermost sclera. Circumferential fibers were directly adjacent to the canal and most prevalent in the outermost, posterior sclera. Interweaving fibers were found throughout the sclera thickness. Across all species, median anisotropy in the radial, circumferential, and interweaving regions were 0.95, 0.96, and 0.28, respectively. Conclusions: Regions of radial, circumferential, and interweaving fibers occur in the posterior pole sclera of human, monkey, pig, sheep, cow, and goat eyes. The consistency across species in scleral architecture suggests that they are primary organizational components whose functions should be better understood.

Thin Lamina Cribrosa Beams Have Different Collagen Microstructure Than Thick Beams.

Purpose: To compare the collagen microstructural crimp characteristics between thin and thick lamina cribrosa (LC) beams. Methods: Seven eyes from four sheep were fixed at 5 mm Hg IOP in 10% formalin. For each eye, one to three coronal cryosections through the LC were imaged with polarized light microscopy and analyzed to visualize the LC and determine collagen fiber microstructure. For every beam, we measured its width and three characteristics of the crimp of its collagen fibers: waviness, tortuosity, and amplitude. Linear mixed effects models were used to test whether crimp characteristics were associated with the LC beam width. Results: For each eye and over all the eyes, LC beam width was positively associated with crimp waviness and tortuosity, and negatively associated with crimp amplitude (P's < 0.0001). Thin beams, average width 13.11 µm, had average (SD) waviness, tortuosity, and amplitude of 0.27 (0.17) radians, 1.017 (0.028) and 1.88 (1.41) µm, respectively. For thick beams, average width 26.10 µm, these characteristics were 0.33 (0.18) radians, 1.025 (0.037) and 1.58 (1.36) µm, respectively. Conclusions: Our results suggest heterogeneity in LC beam mechanical properties. Thin beams were less wavy than their thicker counterparts, suggesting that thin beams may stiffen at lower IOP than thick beams. This difference may allow thin beams to support similar amounts of IOP-induced force as thicker beams, thus providing a similar level of structural support to the axons at physiologic IOP, despite the differences in width. Measurements of beam-level mechanical properties are needed to confirm these predictions.

Spatial Patterns and Age-Related Changes of the Collagen Crimp in the Human Cornea and Sclera.

Purpose: Collagen is the main load-bearing component of the eye, and collagen crimp is a critical determinant of tissue mechanical behavior. We test the hypothesis that collagen crimp morphology varies over the human cornea and sclera and with age. Methods: We analyzed 42 axial whole-globe sections from 20 normal eyes of 20 human donors, ranging in age from 0.08 (1 month) to 97 years. The sections were imaged using polarized light microscopy to obtain µm-scale fiber bundle/lamellae orientation from two corneal and six scleral regions. Crimp morphology was quantified through waviness, tortuosity, and amplitude. Results: Whole-globe median waviness, tortuosity, and amplitude were 0.127 radians, 1.002, and 0.273 µm, respectively. These parameters, however, were not uniform over the globe, instead exhibiting distinct, consistent patterns. All crimp parameters decreased significantly with age, with significantly different age-related decreases between regions. The crimp morphology of the limbus changed the most drastically with age, such that it had the largest crimp in neonates, and among the smallest in the elderly. Conclusions: Age-related decreases in crimp parameters are likely one of the mechanisms underlying age-related stiffening of the sclera and cornea, potentially influencing sensitivity to IOP. Further work is needed to determine the biomechanical implications of the crimp patterns observed. The comparatively large changes in the crimp morphology of the limbus, especially in the early years of life, suggest that crimp in this region may play a role in eye development, although the exact nature of this is unclear.