Predatory bacteria can reduce Pseudomonas aeruginosa induced corneal perforation and proliferation in a rabbit keratitis model.

PURPOSE: Pseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. METHODS: Rabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic ΔlasR mutant and co-injected with PBS or B. bacteriovorus. After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. RESULTS: We observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n = 24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n = 25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The ΔlasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus. CONCLUSION: These studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.

Predatory Bacteria can Reduce Pseudomonas aeruginosa Induced Corneal Perforation and Proliferation in a Rabbit Keratitis Model.

Purpose: Pseudomonas aeruginosa keratitis is a severe ocular infection that can lead to perforation of the cornea. In this study we evaluated the role of bacterial quorum sensing in generating corneal perforation and bacterial proliferation and tested whether co-injection of the predatory bacteria Bdellovibrio bacteriovorus could alter the clinical outcome. P. aeruginosa with lasR mutations were observed among keratitis isolates from a study collecting samples from India, so an isogenic lasR mutant strain of P. aeruginosa was included. Methods: Rabbit corneas were intracorneally infected with P. aeruginosa strain PA14 or an isogenic Δ lasR mutant and co-injected with PBS or B. bacteriovorus . After 24 h, eyes were evaluated for clinical signs of infection. Samples were analyzed by scanning electron microscopy, optical coherence tomography, sectioned for histology, and corneas were homogenized for CFU enumeration and for inflammatory cytokines. Results: We observed that 54% of corneas infected by wild-type PA14 presented with a corneal perforation (n=24), whereas only 4% of PA14 infected corneas that were co-infected with B. bacteriovorus perforate (n=25). Wild-type P. aeruginosa proliferation was reduced 7-fold in the predatory bacteria treated eyes. The Δ lasR mutant was less able to proliferate compared to the wild-type, but was largely unaffected by B. bacteriovorus . Conclusion: These studies indicate a role for bacterial quorum sensing in the ability of P. aeruginosa to proliferate and cause perforation of the rabbit cornea. Additionally, this study suggests that predatory bacteria can reduce the virulence of P. aeruginosa in an ocular prophylaxis model.

Spatial distribution and network morphology of epicardial, endocardial, interstitial, and Purkinje cell-associated elastin fibers in porcine left ventricle.

Cardiac extracellular matrices (ECM) play crucial functional roles in cardiac biomechanics. Previous studies have mainly focused on collagen, the major structural ECM in heart wall. The role of elastin in cardiac mechanics, however, is poorly understood. In this study, we investigated the spatial distribution and microstructural morphologies of cardiac elastin in porcine left ventricles. We demonstrated that the epicardial elastin network had location- and depth-dependency, and the overall epicardial elastin fiber mapping showed certain correlation with the helical heart muscle fiber architecture. When compared to the epicardial layer, the endocardial layer was thicker and has a higher elastin-collagen ratio and a denser elastin fiber network; moreover, the endocardial elastin fibers were finer and more wavy than the epicardial elastin fibers, all suggesting various interface mechanics. The myocardial interstitial elastin fibers co-exist with the perimysial collagen to bind the cardiomyocyte bundles; some of the interstitial elastin fibers showed a locally aligned, hinge-like structure to connect the adjacent cardiomyocyte bundles. This collagen-elastin combination reflects an optimal design in which the collagen provides mechanical strength and elastin fibers facilitate recoiling during systole. Moreover, cardiac elastin fibers, along with collagen network, closely associated with the Purkinje cells, indicating that this ECM association could be essential in organizing cardiac Purkinje cells into "fibrous" and "branching" morphologies and serving as a protective feature when Purkinje fibers experience large deformations in vivo. In short, our observations provide a structural basis for future in-depth biomechanical investigations and biomimicking of this long-overlooked cardiac ECM component.

Investigating the Transient Regenerative Potential of Cardiac Muscle Using a Neonatal Pig Partial Apical Resection Model.

Researchers have shown that adult zebrafish have the potential to regenerate 20% of the ventricular muscle within two months of apex resection, and neonatal mice have the capacity to regenerate their heart after apex resection up until day 7 after birth. The goal of this study was to determine if large mammals (porcine heart model) have the capability to fully regenerate a resected portion of the left ventricular apex during the neonatal stage, and if so, how long the regenerative potential persists. A total of 36 piglets were divided into the following groups: 0-day control and surgical groups and seven-day control and surgical groups. For the apex removal groups, each piglet was subjected to a partial wall thickness resection (~30% of the ventricular wall thickness). Heart muscle function was assessed via transthoracic echocardiograms; the seven-day surgery group experienced a decrease in ejection fraction and fractional shortening. Upon gross necropsy, for piglets euthanized four weeks post-surgery, all 0-day-old hearts showed no signs of scarring or any indication of the induced injury. Histological analysis confirmed that piglets in the 0-day surgery group exhibited various degrees of regeneration, with half of the piglets showing full regeneration and the other half showing partial regeneration. However, each piglet in the seven-day surgery group demonstrated epicardial fibrosis along with moderate to severe dissecting interstitial fibrosis, which was accompanied by an abundant collagenous extracellular matrix as the result of a scar formation in the resection site. Histology of one 0-day apex resection piglet (briefly lain on and accidentally killed by the mother sow three days post-surgery) revealed dense, proliferative mesenchymal cells bordering the fibrin and hemorrhage zone and differentiating toward immature cardiomyocytes. We further examined the heart explants at 5-days post-surgery (5D PO) and 1-week post-surgery (1W PO) to assess the repair progression. For the 0-day surgery piglets euthanized at 5D PO and 1W PO, half had abundant proliferating mesenchymal cells, suggesting active regeneration, while the other half showed increased extracellular collagen. The seven-day surgery piglets euthanized at 5D PO, and 1W PO showed evidence of greatly increased extracellular collagen, while some piglets had proliferating mesenchymal cells, suggesting a regenerative effort is ongoing while scar formation seems to predominate. In short, our qualitative findings suggest that the piglets lose the full myocardial regenerative potential by 7 days after birth, but greatly preserve the regenerative potential within 1 day post-partum.

Eye-specific 3D modeling of factors influencing oxygen concentration in the lamina cribrosa.

Our goal was to identify the factors with the strongest influence on the minimum lamina cribrosa (LC) oxygen concentration as potentially indicative of conditions increasing hypoxia risk. Because direct measurement of LC hemodynamics and oxygenation is not yet possible, we developed 3D eye-specific LC vasculature models. The vasculature of a normal monkey eye was perfusion-labeled post-mortem. Serial cryosections through the optic nerve head were imaged using fluorescence and polarized light microscopy to visualize the vasculature and collagen, respectively. The vasculature within a 450 µm-thick region containing the LC - identified from the collagen, was segmented, skeletonized, and meshed for simulations. Using Monte Carlo sampling, 200 vascular network models were generated with varying vessel diameter, neural tissue oxygen consumption rate, inflow hematocrit, and blood pressures (arteriole, venule, anterior boundary, and posterior boundary). Factors were varied over ranges of baseline ±20% with uniform probability. For each model we first obtained the blood flow, and from this the neural tissue oxygen concentration. ANOVA was used to identify the factors with the strongest influence on the minimum (10th percentile) oxygen concentration in the LC. The three most influential factors were, in ranked order, vessel diameter, neural tissue oxygen consumption rate, and arteriole pressure. There was a strong interaction between vessel diameter and arteriole pressure whereby the impact of one factor was larger when the other factor was small. Our results show that, for the eye analyzed, conditions that reduce vessel diameter, such as vessel compression due to elevated intraocular pressure or gaze-induced tissue deformation, may particularly contribute to decreased LC oxygen concentration. More eyes must be analyzed before generalizing.

A Workflow for Three-Dimensional Reconstruction and Quantification of the Monkey Optic Nerve Head Vascular Network.

A comprehensive characterization of the three-dimensional (3D) vascular network of the optic nerve head (ONH) is critical to understanding eye physiology and pathology. Current in vivo imaging technologies, however, do not have simultaneous high spatial resolution and imaging depth to resolve the small vessels deep within the ONH. We describe a workflow for the 3D reconstruction and quantitative morphological analysis of the ONH vasculature. The vessels of a normal monkey ONH were perfusion labeled. Serial cryosections of the ONH were imaged using fluorescence microscopy (FM) and instant polarized light microscopy (IPOL) to visualize the labeled vessels and label-free collagen, respectively. The IPOL images were registered and used to form a stack of FM images from which the vessels were segmented and skeletonized to reconstruct the 3D vascular network. The network consisted of 12,966 vessel segments, 7989 branching points, and 1100 terminal points at the boundaries. For each vessel segment, we measured its length, tortuosity, inclination (θ), and polar orientation (φ). The length followed a lognormal distribution, whereas the distribution of the tortuosity followed an exponential decay. The vessels were mainly oriented toward the coronal plane (θ = 90 deg). For orientation, there were nearly as many vessels aligned circumferentially (φ = 90 deg) and radially (φ = 0 deg). Our results demonstrate the workflow for 3D eye-specific reconstruction and quantification of the monkey ONH vascular network. This is a critical first step to analyze the blood flow and oxygenation within the ONH, which will help understand the role of vascular dysfunction in glaucoma.

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.

Biomechanical properties of acellular scar ECM during the acute to chronic stages of myocardial infarction.

After myocardial infarction (MI), the infarcted tissue undergoes dynamic and time-dependent changes. Previous knowledge on MI biomechanical alterations has been obtained by studying the explanted scar tissues. In this study, we decellularized MI scar tissue and characterized the biomechanics of the obtained pure scar ECM. By thoroughly removing the cellular content in the MI scar tissue, we were able to avoid its confounding effects. Rat MI hearts were obtained from a reliable and reproducible model based on permanent left coronary artery ligation (PLCAL). MI heart explants at various time points (15 min, 1 week, 2 weeks, 4 weeks, and 12 weeks) were subjected to decellularization with 0.1% sodium dodecyl sulfate solution for ~1-2 weeks to obtain acellular scar ECM. A biaxial mechanical testing system was used to characterize the acellular scar ECM under physiologically relevant loading conditions. After decellularization, large decrease in wall thickness was observed in the native heart ECM and 15 min scar ECM, implying the collapse of cardiomyocyte lacunae after removal of heart muscle fibers. For scar ECM 1 week, 2 weeks, and 4 weeks post infarction, the decrease in wall thickness after decellularization was small. For scar ECM 12 weeks post infarction, the reduction amount of wall thickness due to decellularization was minimal. We found that the scar ECM preserved the overall mechanical anisotropy of the native ventricle wall and MI scar tissue, in which the longitudinal direction is more extensible. Acellular scar ECM from 15 min to 12 weeks post infarction showed an overall stiffening trend in biaxial behavior, in which longitudinal direction was mostly affected and manifested with a decreased extensibility and increased modulus. This reduction trend of longitudinal extensibility also led to a decreased anisotropy index in the scar ECM from the acute to chronic stages of MI. The post-MI change in biomechanical properties of the scar ECM reflected the alterations of collagen fiber network, confirmed by the histology of scar ECM. In short, the reported structure-property relationship reveals how scar ECM biophysical properties evolve from the acute to chronic stages of MI. The obtained information will help establish a knowledge basis about the dynamics of scar ECM to better understand post-MI cardiac remodeling.

Instant polarized light microscopy for imaging collagen microarchitecture and dynamics.

Collagen fibers are a primary load-bearing component of connective tissues and are therefore central to tissue biomechanics and pathophysiology. Understanding collagen architecture and behavior under dynamic loading requires a quantitative imaging technique with simultaneously high spatial and temporal resolutions. Suitable techniques are thus rare and often inaccessible. In this study, we present instant polarized light microscopy (IPOL), in which a single snapshot image encodes information on fiber orientation and retardance, thus fulfilling the requirement. We utilized both simulation and experimental data from collagenous tissues of chicken tendon, sheep eye, and porcine heart to evaluate the effectiveness of IPOL as a quantitative imaging technique. We demonstrate that IPOL allows quantitative characterization of micron-scale collagen fiber architecture at full camera frame rates (156 frames/second herein).

So-Called Lamina Cribrosa Defects May Mitigate IOP-Induced Neural Tissue Insult.

Purpose: The prevailing theory about the function of lamina cribrosa (LC) connective tissues is that they provide structural support to adjacent neural tissues. Missing connective tissues would compromise this support and therefore are regarded as "LC defects", despite scarce actual evidence of their role. We examined how so-called LC defects alter IOP-related mechanical insult to the LC neural tissues. Methods: We built numerical models incorporating LC microstructure from polarized light microscopy images. To simulate LC defects of varying sizes, individual beams were progressively removed. We then compared intraocular pressure (IOP)-induced neural tissue deformations between models with and without defects. To better understand the consequences of defect development, we also compared neural tissue deformations between models with partial and complete loss of a beam. Results: The maximum stretch of neural tissues decreased non-monotonically with defect size. Maximum stretch in the model with the largest defect decreased by 40% in comparison to the model with no defects. Partial loss of a beam increased the maximum stretch of neural tissues in its adjacent pores by 162%, compared with 63% in the model with complete loss of a beam. Conclusions: Missing LC connective tissues can mitigate IOP-induced neural tissue insult, suggesting that the role of the LC connective tissues is more complex than simply fortifying against IOP. The numerical models further predict that partial loss of a beam is biomechanically considerably worse than complete loss of a beam, perhaps explaining why defects have been reported clinically but partial beams have not.

Lamina Cribrosa Capillaries Straighten as Intraocular Pressure Increases.

Purpose: The purpose of this study was to visualize the lamina cribrosa (LC) capillaries and collagenous beams, measure capillary tortuosity (path length over straight end-to-end length), and determine if capillary tortuosity changes when intraocular pressure (IOP) increases. Methods: Within 8 hours of sacrifice, 3 pig heads were cannulated via the external ophthalmic artery, perfused with PBS to remove blood, and then perfused with a fluorescent dye to label the capillaries. The posterior pole of each eye was mounted in a custom-made inflation chamber for control of IOP with simultaneous imaging. Capillaries and collagen beams were visualized with structured light illumination enhanced imaging at IOPs from 5 to 50 mm Hg at each 5 mm Hg increment. Capillary tortuosity was measured from the images and paired two-sample t-tests were used to assess for significant changes in relation to changes in IOP. Results: Capillaries were highly tortuous at 15 mm Hg (up to 1.45). In all but one eye, tortuosity decreased significantly as IOP increased from 15 to 25 mm Hg (P < 0.01), and tortuosity decreased significantly in every eye as IOP increased from 15 to 40 mm Hg (P < 0.01). In only 16% of capillaries, tortuosity increased with elevated IOP. Capillaries had a surprisingly different topology from the collagen beams. Conclusions: Although high capillary tortuosity is sometimes regarded as potentially problematic because it can reduce blood flow, LC capillary tortuosity may provide slack that mitigates against reduced flow and structural damage caused by excessive stretch under elevated IOP. We speculate that low capillary tortuosity could be a risk factor for damage under high IOP.

Collagen fiber interweaving is central to sclera stiffness.

The mechanical properties of the microstructural components of sclera are central to eye physiology and pathology. Because these parameters are extremely difficult to measure directly, they are often estimated using inverse-modeling matching deformations of macroscopic samples measured experimentally. Although studies of sclera microstructure show collagen fiber interweaving, current models do not account for this interweaving or the resulting fiber-fiber interactions, which might affect parameter estimates. Our goal was to test the hypothesis that constitutive parameters estimated using inverse modeling differ if models account for fiber interweaving and interactions. We developed models with non-interweaving or interweaving fibers over a wide range of volume fractions (36-91%). For each model, we estimated fiber stiffness using inverse modeling matching biaxial experimental data of human sclera. We found that interweaving increased the estimated fiber stiffness. When the collagen volume fraction was 64% or less, the stiffness of interweaving fibers was about 1.25 times that of non-interweaving fibers. For higher volume fractions, the ratio increased substantially, reaching 1.88 for a collagen volume fraction of 91%. Simulating a model (interweaving/non-interweaving) using the fiber stiffness estimated from the other model produced substantially different behavior, far from that observed experimentally. These results show that estimating microstructural component mechanical properties is highly sensitive to the assumed interwoven/non-interwoven architecture. Moreover, the results suggest that interweaving plays an important role in determining the structural stiffness of sclera, and potentially of other soft tissues in which the collagen fibers interweave. STATEMENT OF SIGNIFICANCE: The collagen fibers of sclera are interwoven, but numerical models do not account for this interweaving or the resulting fiber-fiber interactions. To determine if interweaving matters, we examined the differences in the constitutive model parameters estimated using inverse modeling between models with interweaving and non-interweaving fibers. We found that the estimated stiffness of the interweaving fibers was up to 1.88 times that of non-interweaving fibers, and that the estimate increased with collagen volume fraction. Our results suggest that fiber interweaving is a fundamental characteristic of connective tissues, additional to anisotropy, density and orientation. Better characterization of interweaving, and of its mechanical effects is likely central to understanding microstructure and biomechanics of sclera and other soft tissues.

Heart valve tissue-derived hydrogels: Preparation and characterization of mitral valve chordae, aortic valve, and mitral valve gels.

Heart valve (HV) diseases are among the leading causes of death and continue to threaten public health worldwide. The current clinical options for HV replacement include mechanical and biological prostheses. However, an ongoing problem with current HV prostheses is their failure to integrate with the host tissue and their inability grow and remodel within the body. Tissue engineered heart valves (TEHVs) are a promising solution to these problems, as they are able to grow and remodel somatically with the rest of the body. Recently, decellularized HVs have demonstrated great potential as valve replacements because they are tissue specific, but recellularization is still a challenge due to the dense HV extracellular matrix (ECM) network. In this proof-of-concept work, we decellularized porcine mitral valve chordae, aortic valve leaflets, and mitral valve leaflets and processed them into injectable hydrogels that could accommodate any geometry. While the three valvular ECMs contained various amounts of collagen, they displayed similar glycosaminoglycan contents. The hydrogels had similar nanofibrous structures and gelation kinetics with various compressive strengths. When encapsulated with NIH 3 T3 fibroblasts, all the hydrogels supported cell survivals up to 7 days. Decellularized HV ECM hydrogels may show promising potential HV tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1732-1740, 2019.

Structured polarized light microscopy for collagen fiber structure and orientation quantification in thick ocular tissues.

Collagen is a major constituent of the eye and understanding its architecture and biomechanics is critical to preserve and restore vision. We, recently, demonstrated polarized light microscopy (PLM) as a powerful technique for measuring properties of the collagen fibers of the eye, such as spatial distribution and orientation. Our implementation of PLM, however, required sectioning the tissues for imaging using transmitted light. This is problematic because it limits analysis to thin sections. This is not only slow, but precludes study of dynamic events such as pressure-induced deformations, which are central to the role of collagen. We introduce structured polarized light microscopy (SPLM), an imaging technique that combines structured light illumination with PLM to allow imaging and measurement of collagen fiber properties in thick ocular tissues. Using pig and sheep eyes, we show that SPLM rejects diffuse background light effectively in thick tissues, significantly enhancing visualization of optic nerve head (ONH) structures, such as the lamina cribrosa, and improving the accuracy of the collagen fiber orientation measurements. Further, we demonstrate the integration of SPLM with an inflation device to enable direct visualization, deformation tracking, and quantification of collagen fibers in ONHs while under controlled pressure.

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.

Quantitative Analysis of Tissue Damage Evolution in Porcine Liver With Interrupted Mechanical Testing Under Tension, Compression, and Shear.

In this study, the damage evolution of liver tissue was quantified at the microstructural level under tensile, compression, and shear loading conditions using an interrupted mechanical testing method. To capture the internal microstructural changes in response to global deformation, the tissue samples were loaded to different strain levels and chemically fixed to permanently preserve the deformed tissue geometry. Tissue microstructural alterations were analyzed to quantify the accumulated damages, with damage-related parameters such as number density, area fraction, mean area, and mean nearest neighbor distance (NND). All three loading states showed a unique pattern of damage evolution, in which the damages were found to increase in number and size, but decrease in NND as strain level increased. To validate the observed damage features as true tissue microstructural damages, more samples were loaded to the above-mentioned strain levels and then unloaded back to their reference state, followed by fixation. The most major damage-relevant features at higher strain levels remained after the release of the external loading, indicating the occurrence of permanent inelastic deformation. This study provides a foundation for future structure-based constitutive material modeling that can capture and predict the stress-state dependent damage evolution in liver tissue.

Polarized light microscopy for 3-dimensional mapping of collagen fiber architecture in ocular tissues.

Collagen fibers play a central role in normal eye mechanics and pathology. In ocular tissues, collagen fibers exhibit a complex 3-dimensional (3D) fiber orientation, with both in-plane (IP) and out-of-plane (OP) orientations. Imaging techniques traditionally applied to the study of ocular tissues only quantify IP fiber orientation, providing little information on OP fiber orientation. Accurate description of the complex 3D fiber microstructures of the eye requires quantifying full 3D fiber orientation. Herein, we present 3dPLM, a technique based on polarized light microscopy developed to quantify both IP and OP collagen fiber orientations of ocular tissues. The performance of 3dPLM was examined by simulation and experimental verification and validation. The experiments demonstrated an excellent agreement between extracted and true 3D fiber orientation. Both IP and OP fiber orientations can be extracted from the sclera and the cornea, providing previously unavailable quantitative 3D measures and insight into the tissue microarchitecture. Together, the results demonstrate that 3dPLM is a powerful imaging technique for the analysis of ocular tissues.

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.

Cardiac findings in Quarter Horses with heritable equine regional dermal asthenia.

OBJECTIVE To compare biomechanical and histologic features of heart valves and echocardiographic findings between Quarter Horses with and without heritable equine regional dermal asthenia (HERDA). DESIGN Prospective case-control study. ANIMALS 41 Quarter Horses. PROCEDURES Ultimate tensile strength (UTS) of aortic and mitral valve leaflets was assessed by biomechanical testing in 5 horses with HERDA and 5 horses without HERDA (controls). Histologic evaluation of aortic and mitral valves was performed for 6 HERDA-affected and 3 control horses. Echocardiography was performed in 14 HERDA-affected and 11 control horses. Biomechanical data and echocardiographic variables of interest were compared between groups by statistical analyses, RESULTS Mean values for mean and maximum UTS of heart valves were significantly lower in HERDA-affected horses than in controls. Blood vessels were identified in aortic valve leaflets of HERDA-affected but not control horses. Most echocardiographic data did not differ between groups. When the statistical model for echocardiographic measures was controlled for body weight, mean and maximum height and width of the aorta at the valve annulus in short-axis images were significantly associated with HERDA status and were smaller for affected horses. CONCLUSIONS AND CLINICAL RELEVANCE Lower UTS of heart valves in HERDA-affected horses, compared with those of control horses, supported that tissues other than skin with high fibrillar collagen content are abnormal in horses with HERDA. Lack of significant differences in most echocardiographic variables between affected and control horses suggested that echocardiography may not be useful to detect a substantial loss of heart valve tensile strength. Further investigation is warranted to confirm these findings. Studies in horses with HERDA may provide insight into cardiac abnormalities in people with collagen disorders.