Ablation of mpeg+ Macrophages Exacerbates mfrp-Related Hyperopia.

Purpose: Proper refractive development of the eye, termed emmetropization, is critical for focused vision and is impacted by both genetic determinants and several visual environment factors. Improper emmetropization caused by genetic variants can lead to congenital hyperopia, which is characterized by small eyes and relatively short ocular axial length. To date, variants in only four genes have been firmly associated with human hyperopia, one of which is MFRP. Zebrafish mfrp mutants also have hyperopia and, similar to reports in mice, exhibit increased macrophage recruitment to the retina. The goal of this research was to examine the effects of macrophage ablation on emmetropization and mfrp-related hyperopia. Methods: We utilized a chemically inducible, cell-specific ablation system to deplete macrophages in both wild-type and mfrp mutant zebrafish. Spectral-domain optical coherence tomography was then used to measure components of the eye and determine relative refractive state. Histology, immunohistochemistry, and transmission electron microscopy were used to further study the eyes. Results: Although macrophage ablation does not cause significant changes to the relative refractive state of wild-type zebrafish, macrophage ablation in mfrp mutants significantly exacerbates their hyperopic phenotype, resulting in a relative refractive error 1.3 times higher than that of non-ablated mfrp siblings. Conclusions: Genetic inactivation of mfrp leads to hyperopia, as well as abnormal accumulation of macrophages in the retina. Ablation of the mpeg1-positive macrophage population exacerbates the hyperopia, suggesting that macrophages may be recruited in an effort help preserve emmetropization and ameliorate hyperopia.

Declines in PDE4B activity promote myopia progression through downregulation of scleral collagen expression.

Myopia is the most common cause of a visual refractive error worldwide. Cyclic adenosine monophosphate (cAMP)-linked signaling pathways contribute to the regulation of myopia development, and increases in cAMP accumulation promote myopia progression. To pinpoint the underlying mechanisms by which cAMP modulates myopia progression, we performed scleral transcriptome sequencing analysis in form-deprived mice, a well-established model of myopia development. Form deprivation significantly inhibited the expression levels of genes in the cAMP catabolic pathway. Quantitative real-time polymerase chain reaction analysis validated that the gene expression level of phosphodiesterase 4B (PDE4B), a cAMP hydrolase, was downregulated in form-deprived mouse eyes. Under visually unobstructed conditions, loss of PDE4B function in Pde4b-knockout mice increased the myopic shift in refraction, -3.661 ± 1.071 diopters, more than that in the Pde4b-wildtype littermates (P < 0.05). This suggests that downregulation and inhibition of PDE4B gives rise to myopia. In guinea pigs, subconjunctival injection of rolipram, a selective inhibitor of PDE4, led to myopia in normal eyes, and it also enhanced form-deprivation myopia (FDM). Subconjunctival injection of dibutyryl-cyclic adenosine monophosphate, a cAMP analog, induced only a myopic shift in the normal visually unobstructed eyes, but it did not enhance FDM. As myopia developed, axial elongation occurred during scleral remodeling that was correlated with changes in collagen fibril thickness and distribution. The median collagen fibril diameter in the FDM + rolipram group, 55.09 ± 1.83 nm, was thinner than in the FDM + vehicle group, 59.33 ± 2.06 nm (P = 0.011). Thus, inhibition of PDE4 activity with rolipram thinned the collagen fibril diameter relative to the vehicle treatment in form-deprived eyes. Rolipram also inhibited increases in collagen synthesis induced by TGF-ß2 in cultured human scleral fibroblasts. The current results further support a role for PDE enzymes such as PDE4B in the regulation of normal refractive development and myopia because either loss or inhibition of PDE4B function increased myopia and FDM development through declines in the scleral collagen fibril diameter.

Effect of Regulating the Expression of HSP47 on Collagen Metabolism in Scleral Fibroblasts.

PURPOSE: To explore the effect of heat shock protein 47 (HSP 47) on collagen (types I, III, and V) metabolism in scleral fibroblasts. METHODS: Scleral fibroblasts with over- or low-expression of HSP 47 were constructed by plasmid transfection. The mRNA and intracellular proteins expression of HSP 47, collagen (types I, III, and V) and α-smooth muscle actin (α-SMA) were detected by quantitative real-time polymerase chain reaction and western blot. The proteins expression of collagen (types I, III, and V), matrix metalloproteinase 2 (MMP-2), and tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP-1 and -2) in extracellular matrix (ECM) were detected by ELISA. The migration and proliferation activities of cells were detected by scratch-wound assay and MTS. The internal structure of scleral fibroblasts was observed by transmission electron microscopy (TEM). RESULTS: The results obtained demonstrated significant increases in the expression of the mRNA and protein expression of collagen I in HSP47 up-regulated cells. Overexpression of HSP 47 promotes the expression of α-SMA and cell migration. Down-regulated expression of HSP 47 results in decreased mRNA and protein expression of collagen. Low expression of HSP 47 significantly inhibits cell migration and proliferation, and affects the internal structure of cells. CONCLUSION: HSP 47 affects collagen metabolism in scleral fibroblasts. It appears to promote the synthesis and secretion of collagen I as well as inhibit degradation.

Safety and Long-term Scleral Biomechanical Stability of Rhesus Eyes after Scleral Cross-linking by Blue Light.

Purpose: To assess the safety and long-term scleral biomechanical stability of rhesus eyes after blue light scleral CXL by investigating the biomechanical and microstructural changes.Methods: Seven rhesus monkeys (14 eyes) were observed in this study. All right eyes received blue light scleral CXL at the superior temporal equatorial sclera, and the left eyes served as controls. Biological ocular parameters were followed up to 1 year after scleral CXL. Stress-strain measurements of three rhesus sclera were measured, three rhesus retinas were examined histologically by H&E and TUNEL staining. And the microstructure of both the sclera and retina were observed by transmission electron microscopy at 1 year.Results: As for the retinal thickness, choroidal thickness, flow density of retinal superficial vascular networks and flash electroretinography (f-ERG) results, no significant differences were observed between the paired eyes at 1 year (P >.05). At the same time, the scleral collagen fibril distribution was much tighter, and the scleral biomechanical properties were significantly increased in the experimental eyes. However, apoptotic cells and retinal ultrastructural changes could still be found in the retina of the experimental eyes.Conclusion: This study demonstrates that blue light scleral CXL could effectively increase the scleral stiffness of the rhesus eye for at least 1 year, but ultrastructural change was still observed in the retina of scleral CXL eye. Therefore, the long-term intraocular safety of the blue light scleral CXL technique for preventing myopia progression should be investigated further.

3D-Reconstruction of the human conventional outflow system by ribbon scanning confocal microscopy.

PURPOSE: The risk for glaucoma is driven by the microanatomy and function of the anterior segment. We performed a computation-intense, high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM) of the outflow tract of two human eyes. We hypothesized this would reveal important species differences when compared to existing data of porcine eyes, an animal that does not spontaneously develop glaucoma. METHODS: After perfusing two human octogenarian eyes with lectin-fluorophore conjugate and optical clearance with benzyl alcohol benzyl benzoate (BABB), anterior segments were scanned by RSCM and reconstructed in 3D for whole-specimen rendering. Morphometric analyses of the outflow tract were performed for the trabecular meshwork (TM), limbal, and perilimbal outflow structures and compared to existing porcine data. RESULTS: RSCM provided high-resolution data for IMARIS-based surface reconstruction of outflow tract structures in 3D. Different from porcine eyes with an abundance of highly interconnected, narrow, and short collector channels (CCs), human eyes demonstrated fewer CCs which had a 1.5x greater cross-sectional area (CSA) and 2.6x greater length. Proximal CC openings at the level of Schlemm's canal (SC) had a 1.3x larger CSA than distal openings into the scleral vascular plexus (SVP). CCs were 10.2x smaller in volume than the receiving SVP vessels. Axenfeld loops, projections of the long ciliary nerve, were also visualized. CONCLUSION: In this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes. Human CCs spanned several clock-hours and were larger than in porcine eyes. These species differences may point to factors downstream of the TM that increase our vulnerability to glaucoma.

Localization of Tfap2ß, Casq2, Penk, Zic1, and Zic3 Expression in the Developing Retina, Muscle, and Sclera of the Embryonic Mouse Eye.

Optic development involves sequential interactions between several different tissue types, including the overlying ectoderm, adjacent mesoderm, and neural crest mesenchyme and the neuroectoderm. In an ongoing expression screen, we identified that Tfap2ß, Casq2, Penk, Zic1, and Zic3 are expressed in unique cell types in and around the developing eye. Tfap2ß, Zic1, and Zic3 are transcription factors, Casq2 is a calcium binding protein and Penk is a neurotransmitter. Tfap2ß, Zic1, and Zic3 have reported roles in brain and craniofacial development, while Casq2 and Penk have unknown roles. These five genes are expressed in the major tissue types in the eye, including the muscles, nerves, cornea, and sclera. Penk expression is found in the sclera and perichondrium. At E12.5 and E15.5, the extra-ocular muscles express Casq2, the entire neural retina expresses Zic1, and Zic3 is expressed in the optic disk and lip of the optic cup. The expression of Tfap2ß expanded from corneal epithelium to the neural retina between E12.5 to E15.5. These genes are expressed in similar domains as Hedgehog (Gli1, and Ptch1) and the Wnt (Lef1) pathways. The expression patterns of these five genes warrant further study to determine their role in eye morphogenesis.

Effect of chronic topical latanoprost on the sclera and lamina cribrosa of form-deprived myopic Guinea pigs.

The purpose of this study was to investigate the effects of latanoprost, an ocular hypotensive prostaglandin analog, on scleral collagen fibers and laminar pores in myopic guinea pigs. Young guinea pigs underwent monocular form deprivation (FD; white plastic diffusers) from 14-days of age for 10-weeks. After the first week, FD eyes also received daily topical A) latanoprost (Lat, 0.005%, n = 5) or B) artificial tears (AT; n = 5). At the end of the treatment period, animals were sacrificed, eyes enucleated and optic nerve heads (ONH) excised to include a 4 mm diameter ring of surrounding sclera for scanning electron microscopy (SEM), and an additional 6 mm ring of sclera surrounding the ONH was excised for transmission electron microscopy (TEM). For SEM, ONH samples were first immersed in 0.2M NaOH for 30 h to isolate the collagenous structures. All samples were stained with osmium tetroxide, dried through an ethanol series and finally subjected to critical point drying before imaging. Image J was used to analyze the dimensions of laminar pores (SEM images) and scleral collagen fibers (TEM images). As previously reported in a related study, latanoprost was effective in inhibiting myopia progression in FD eyes of the guinea pigs. The scleral fibers of FD myopic eyes treated with AT were smaller and more variable in cross-sectional areas compared to untreated (fellow) eyes (mean areas: 0.0059 ±â€¯0.0013 vs. 0.0085 ±â€¯0.002 µm2; p < 0.001), consistent with scleral changes reported for human myopia. In contrast, the scleral fibers of the Lat-treated FD eyes were similar to those of fellow eyes (0.0083 ±â€¯0.002 vs. 0.0078 ±â€¯0.0014 µm2). However, laminar pore size appeared unaffected by either the FD or drug treatments, with no significant difference found between FD eyes and their fellows, for either treatment group. That daily topical latanoprost appeared to protect against myopia-related changes in scleral collagen, rather than exaggerating them, as might be predicted from its known action on the uveoscleral extracellular matrix, lends further support its use for myopia control. In this guinea pig myopia model, the lamina cribrosa appeared unaffected.

Effects of scleral collagen crosslinking with different carbohydrate on chemical bond and ultrastructure of rabbit sclera: Future treatment for myopia progression.

BACKGROUND: Myopia is the most common ocular disorder and is mainly caused by axial elongation of the sclera. If the stiffness of sclera increased, it can inhibit myopia progression. The aim of this study is to compare the effect of the collagen crosslinking with different types and concentrations of carbohydrates on chemical bond and ultrastructural change of rabbit sclera. METHODS: Nine New Zealand white rabbits were treated with five, sequential sub-Tenon injections of 0.15 mL solutions of ribose, sucrose, and glycogen of 0.1, 0.2 and 0.4 M concentration at the right eye over 14 days. Ten weeks after the last injection, the rabbits were sacrificed and chemical bond and ultrastructural changes were compared with those of the untreated left sclera using Raman spectroscopy, atomic force microscopy (AFM), and histology. RESULTS: Raman spectroscopy of the control and cross-linked rabbit sclera tissue revealed different types of collagen interactions. Raman shift of 919 cm-1 (C-C stretching and vibration of the proline ring in collagen) was the highest in ribose, followed by sucrose and glycogen. Total energy intensity was also highest in ribose, followed by sucrose and glycogen, and showed a tendency to increase at higher concentrations. AFM revealed interlocking arrangements of collagen fibrils. The collagen fibril diameter was 105.6 ± 21.2 nm, 109.4 ± 28.8 nm, 113.1 ± 30.8 nm and 137.6 ± 25.3 nm for control group, 0.4 M glycogen, sucrose, and ribose, respectively. Histology indicated increased density of the collagen bundle and no increase in inflammatory cell recruitment compared to control at high concentrations of ribose. CONCLUSIONS: Scleral crosslinking using glycation increased the scleral biomechanical rigidity and these results were particularly pronounced in ribose. Scleral crosslinking using glycation may be a promising method for inhibiting high myopia progression.

Microarchitecture of Schlemm Canal Before and After Cataract Extraction Surgery.

PRECIS: Schlemm canal (SC) expands after cataract extraction (CE), both in the area and in volume by 25% as was measured using enhanced-depth imaging optical coherent tomography (EDI-OCT) in patients before and 1 week after CE. PURPOSE: This study aims to characterize the structural and volume changes on the microstructure of SC in patients before and after uneventful phacoemulsification CE by using EDI-OCT. MATERIALS AND METHODS: Forty-one serial horizontal EDI-OCT B-scans (interval between B-scans, 69 µm) were obtained in the nasal corneoscleral limbus before and 1 week after CE. The structure of aqueous channels, conjunctival blood vessels and iris anatomy in each scan were used as landmarks to select for overlapping scans taken before and following CE. The SC cross-section area was measured in each of the selected scans and SC volume was determined following a 3-dimensional reconstruction. RESULTS: Eleven eyes (6 females and 5 males) were imaged successfully before and after CE. Mean age was 70.54±11.38 years. The mean axial length was 23.10±0.87 mm. After CE, the mean best-corrected visual acuity in logMAR improved from 0.4±0.13 to 0.2±0.13 (P=0.028). There was no significant change in the mean intraocular pressure before and after CE (15.09±1.33 to 15.0±2.16 mm Hg; P=0.39). The mean SC cross-section area increased by 25%, from 4744±376 to 5941±1048 µm (P<0.001). SC volume in the analyzed region increased by 25% from 6,641,473±585,954 to 8,317,909±1,328,809 µm (P<0.001). CONCLUSION: CE expands SC dimensions in healthy eyes. EDI-OCT imaging of SC may prove useful in the evaluation of the SC dimensions in vivo before and after CE.

Morphometric analysis in mouse scleral fibroblasts using focused ion beam/scanning electron microscopy.

The sclera as well as the cornea forms the principal part of the outer fibrous coat of the eye, with a primary function of protecting the intraocular contents and maintaining the shape of the globe. However, the exact morphometric arrangement of scleral fibroblasts remains unclarified. The aim of this study was to observe the three-dimensional structure of the mouse scleral fibroblasts by focused ion beam/scanning electron microscopy (FIB/SEM). Four eyes from C57BL/6J mice were fixed using a mixture of glutaraldehyde and formaldehyde. The sclera was cut out at the equatorial portion and the posterior pole, and postfixed with potassium ferrocyanide, osmium, thiocarbohydrazide, uranyl acetate and lead aspartate. Specimens were then dehydrated and embedded in an epoxy resin. Serial block face images were obtained using FIB/SEM. Three-dimensional image reconstruction and segmentation of the image stack were created using computer software (Amira v6.0.1, FEI). Scleral fibroblasts were arranged in collagenous layers. The cells frequently showed a cellular junction with the neighboring cells and formed cellular networks. Compared with equatorial fibroblasts, there was a more complicated cellular arrangement of the posterior scleral fibroblasts.

Cryopreservation (-20°C) of equine corneoscleral tissue: Microbiological, histological, and ultrastructural study.

OBJECTIVE: To evaluate microbiological, histological, and ultrastructural characteristics of short-term cryopreserved (STC) equine corneoscleral tissue (<1 year), and to compare it with long-term cryopreserved (LTC) tissue (>7 years). ANIMALS STUDIED: Thirty-four healthy equine globes. PROCEDURE: After a decontamination protocol, globes were enucleated and stored at -20°C in broad-spectrum antibiotics. Corneoscleral tissue was evaluated at different storage periods: 1 month-1 year (20 eyes) and 7-9 years (12 eyes). Two eyes were used as controls. Microbiologic study included direct (blood, McConkey, and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopreservation artifacts were evaluated by hematoxylin-eosin. Corneoscleral collagen organization and number of normal and dead keratocytes were established by transmission electron microscopy. RESULTS: All microbiologic direct cultures were negative. Enrichment cultures were positive in 12.5% of corneal and 59.4% of scleral tissues (pcornea  = 0.136; psclera  = 1.000). Cryopreservation artifacts were most commonly observed in LTC tissues (P = 0.002). Normal keratocytes were predominant in STC corneas (STC 60% and LTC 0%) and apoptotic ones in LTC (STC 40% and LTC 90%), whereas necrotic keratocytes were only seen in LTC (LTC 10%) (P = 0.001). No structural differences were detected in collagen organization between STC and LTC (pcornea  = 1.000; psclera  = 0.703). CONCLUSIONS: Cryopreservation of equine corneoscleral tissue did not yield direct bacterial contamination. Apoptosis is the main cause of death of cryopreserved equine keratocytes. Based on the lack of significant structural differences between STC and LTC samples, these cryopreserved tissues could potentially be used for tectonic support for at least 9 years without structural or microbiological impediment.

The pilocarpine-induced ciliary body contraction affects the elastic modulus and collagen of cornea and sclera in early development.

The aim of this study was to investigate the effects of pilocarpine-induced ciliary body constant contraction for a long time period on the elastic modulus and collagen in corneal and scleral tissues in the early developmental stage. Twelve one-month-old New Zealand white rabbits were randomly monocularly treated with pilocarpine to cause ciliary body constant contraction. After 1- and 2-months, the sclera tissues and the eyeballs were obtained to assess the cornea and three regions of the sclera-anterior, equatorial and posterior. The corneal tissues and the three regions of the scleral tissues were cut into strips for elastic modulus measurement using an Instron 5544, the size distribution of collagen fibrils was examined using electron microscopy, the samples were homogenized, and the concentration of hydroxyproline was measured to determine the collagen content. For corneal tissues, long-term pilocarpine-induced ciliary body constant contraction did not affect the elastic modulus and collagen. For scleral tissues, long-term pilocarpine-induced ciliary body constant contraction affected the elastic modulus, diameter of collagen fibrils and collagen content in the equatorial and posterior sclera, however, the anterior sclera were unaffected. These results suggested that pilocarpine-induced ciliary body contraction could affect the scleral structure and lead to deformation of the sclera and eyeball, thereby affecting visual functions function in the process of ocular emmetropization.

In vivo optic nerve head mechanical response to intraocular and cerebrospinal fluid pressure: imaging protocol and quantification method.

This study presents a quantification method for the assessment of the optic nerve head (ONH) deformations of the living human eye under acute intraocular pressure (IOP) elevation and change of cerebrospinal fluid pressure (CSFP) with body position. One eye from a brain-dead organ donor with open-angle glaucoma was imaged by optical coherence tomography angiography during an acute IOP and CSFP elevation test. Volumetric 3D strain was computed by digital volume correlation. With increase in IOP the shear strain consistently increased in both sitting and supine position (p < 0.001). When CSFP was increased at constant IOP by changing body position, a global reduction in the ONH strain was observed (-0.14% p = 0.0264). Strain in the vasculature was significantly higher than in the structural tissue (+0.90%, p = 0.0002). Retinal nerve fiber layer (RNFL) thickness strongly associated (ρ = -0.847, p = 0.008) with strain in the peripapillary sclera (ppScl) but not in the retina (p = 0.433) and lamina (p = 0.611). These initial results show that: CSFP independently to IOP modulates strain in the human ONH; ppScl strains are greater than strains in lamina and retina; strain in the retinal vasculature was higher than in the structural tissue; In this glaucoma eye, higher ppScl strain associated with lower RNFL thickness.

The telopode- and filopode-projecting heterogeneous stromal cells of the human sclera niche.

Telocytes (TCs) are stromal cells defined by the presence of long and slender prolongations (telopodes). They are a biologically and functionally heterogeneous population that has not been previously investigated in the sclera. The purpose of this study is to investigate the presence and characteristics of scleral telocytes through a combined immunohistochemical and transmission electron microscopy (TEM) study using samples from ten adult patients. Stromal cells with a TC-like morphology expressed CD34, CD45, CD105, vimentin and occasionally CD68 but were negative for collagen III, CD31, CD133, and CD146. Conjunctival epithelial cells expressed CD45, CD105, CD146, and vimentin. These phenotypes support a scleral niche with immune TCs and haematopoietic stem cells (HSCs). In TEM, we often found spindle-shaped stromal cells projecting telopodes or filopodes, with extremely long nuclei extended even within those prolongations. We separated these cells into a light subtype, which contained a complete set of organelles, and a dark subtype, consisting of undifferentiated stem/progenitor cells. The light cells contained dense vesicles, Weibel-Palade bodies, and rounded α-granule-like structures. These storage areas for the von Willebrand factor (vWF) are known to express selectins that are critically involved in HSC homing and could also indicate endothelial progenitors. The dark cells were scarcely myoid, populated the episcleral perivascular niches and the scleral stroma, and were equipped with lipid storage areas such as lamellar bodies and lipid droplets (LDs). Previously, unreported intranuclear LDs were found in these cells, which is characteristic of an HSC population. It appears that the human scleral stroma is a niche harbouring TC-like cells with immune and HSC phenotypes, and the mere presence or characteristics of telopodes are not enough to differentiate them.

Telocyte-like cells containing Weibel-Palade bodies in rat lamina fusca.

Telocytes (TCs) are cells with long, thin and moniliform processes called telopodes. These cells have been found in numerous tissues, including the eye choroid and sclera. Lamina fusca (LF), an anatomical structure located at the sclera-choroid junction, has outer fibroblastic lamellae containing cells with long telopodes. The purpose of this study was to evaluate, via transmission electron microscopy, the LF for the presence of endothelial-specific ultrastructural features, such as Weibel-Palade bodies (WPBs), in the residing TCs. We found that the outer fibroblastic layer of LF lacked pigmented cells but contained numerous cells with telopodes. These cells had incomplete or absent basal laminae, were united by focal adhesions and close contacts, and displayed scarce caveolae and shedding vesicles. Within the stromal cells of LF, numerous WPBs in various stages of maturation and vesicular structures, as secretory pods that ensure the exocytosis of WPBs content, were observed. The WPBs content of the cells with telopodes in the LF could indicate either their involvement in vasculogenesis and/or lymphangiogenesis or that they are the P-selectin- and CD63-containing pools that play roles in scleral or choroidal inflammation.

Cryopreservation (-20 °C) of canine corneoscleral tissue: histological, microbiological, and ultrastructural study.

OBJECTIVE: To evaluate microbiological, histological, and ultrastructural characteristics of short-term cryopreserved (STC) canine corneoscleral tissue (<1 year) and to compare it with long-term cryopreserved (LTC) tissue (>6 years). ANIMALS STUDIED: Thirty-six healthy canine globes. PROCEDURE: After a decontamination protocol, globes were enucleated and stored at -20 °C. Corneoscleral tissue was evaluated at different periods: <1 year (20 eyes) and >6 years (12 eyes). Four eyes were used as controls. Microbiologic study included direct (blood, McConkey and Sabouraud agars) and enrichment (brain-heart infusion broth) cultures. Cryopreservation artifacts were evaluated by hematoxylin-eosin. Corneoscleral collagen organization and number of normal and dead keratocytes were established by transmission electron microscopy (TEM). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was also used for keratocyte characterization. RESULTS: Corneal microbial growth was observed in 25% of the direct STC cultures, and in 47.4% and 16.7% of the enriched STC and LTC cultures, respectively. Scleral STC direct cultures were 30% positive, while enrichment cultures were positive in 66.7% and 16.7% of the STC and LTC, respectively (P = 0.011). Cryopreservation artifacts were higher in LTC tissues (P < 0.001). Apoptotic keratocytes were predominant by TEM and TUNEL, in both STC and LTC. Minimal structural differences were detected in collagen organization between STC and LTC. CONCLUSIONS: Cryopreservation of canine corneoscleral tissue seems to reduce bacterial contamination over time. Apoptosis is the main way of death of cryopreserved canine keratocytes. Based on the lack of significant structural differences between STC and LTC samples, these cryopreserved tissues could potentially be used for tectonic support for at least 8 years without structural or microbiological impediment.

Establishment of novel therapy to reduce progression of myopia in rats with experimental myopia by fibroblast transplantation on sclera.

Myopia is one of the most common visual disorders, and is characterized by a progressive axial elongation of the eye. Several methods have been tried to reduce the progression of axial elongation and myopia, but there are still no well-accepted procedures. We hypothesized that transplantation of fibroblasts on the sclera would lead to the synthesis of collagen fibrils on the sclera and reinforce it, and reduce the degree of axial elongation of eyes with form deprivation myopia. To examine this, we developed a form deprivation myopia model in albino Wistar rats and examined the effects of human fibroblasts (hFbs) transplantation on the sclera in the progression of myopia and axial elongation. We found that the form deprivation by eyelid suture induced a myopic shift and axial elongation associated with a thinner sclera and smaller-diameter collagen fibrils in Wistar rats. We also found that the transplanted hFbs synthesized type 1 collagen fibrils on the rat sclera, and these eyes with form deprivation had significantly reduced ocular elongation and myopic shift than the eyes without hFbs transplantation. Some of the synthesized collagen fibrils migrated into the sclera and had a bundle-like appearance and a stripe-like pattern, indicating they had mature characteristics. These findings suggest that the rat sclera was reinforced by the newly synthesized collagen fibrils and the axial elongation was reduced. These results can provide important information for the development of a therapy targeting myopia in humans. Copyright © 2017 John Wiley & Sons, Ltd.

Crosslinking Enzyme Lysyl Oxidase Modulates Scleral Remodeling in Form-Deprivation Myopia.

PURPOSE: Scleral remodeling causes the excessive ocular elongation that underlies myopia. Lysyl oxidase (LOX), a copper-containing amine oxidase, can catalyze collagen and elastin crosslinking. The purpose of this study was to investigate the role of LOX in scleral remodeling in form-deprivation myopia (FDM). METHODS: Seventy-five guinea pigs were randomly divided into five groups as follows: a normal control group, an FDM group, an FDM plus ß-aminopropionitrile (BAPN) group, an FDM plus TGF-ß1 (TGF-ß1) group, and an FDM plus vehicle group. A translucent diffuser was used to induce FDM, and intravitreal injection was used to administer BAPN, TGF-ß1 or vehicle. The scleral LOX and collagen gene and protein levels and the posterior scleral ultrastructure and biomechanics were measured. RESULTS: In the FDM group, both the scleral LOX and collagen gene and protein levels were significantly lower than those in the control eyes. The collagen fibril diameters were significantly decreased in the FDM group compared with the diameters in the control group. A significant decrease in LOX gene and protein expression was observed after BAPN injection, and an increase was observed after TGF-ß1 treatment compared with the levels in the FDM group. Additionally, the scleral collagen fibrils were significantly decreased in the BAPN-treated eyes but increased in the TGF-ß1-treated eyes compared with the FDM eyes. The ultimate stress and Young's modulus of the sclera were lowest in the BAPN group, followed by the FDM group and the TGF-ß1 group. The ultimate strain (%) of the sclera was lowest in the TGF-ß1 group, followed by the FDM group and the BAPN group. CONCLUSION: LOX expression was significantly lowered in myopic sclera. Modulating LOX expression induced a change in both the scleral collagen fibril diameter and the scleral biomechanics. Therefore, LOX may play a key role in the myopia scleral remodeling procedure.

Bulk changes in posterior scleral collagen microstructure in human high myopia.

Purpose: We aimed to characterize any bulk changes in posterior scleral collagen fibril bundle architecture in human eyes with high myopia. Methods: Wide-angle X-ray scattering (WAXS) was employed to map collagen orientation at 0.5 mm × 0.5 mm spatial intervals across the posterior sclera of seven non-myopic human eyes and three eyes with high myopia (>6D of refractive error). At each sampled point, WAXS provided thickness-averaged measures of the angular distribution of preferentially aligned collagen fibrils within the tissue plane and the anisotropic proportion (the ratio of preferentially aligned to total collagen scatter). Results: Non-myopic specimens featured well-conserved microstructural features, including strong uniaxial collagen alignment along the extraocular muscle insertion sites of the mid-posterior sclera and a highly anisotropic annulus of collagen circumscribing the nerve head in the peripapillary sclera. All three myopic specimens exhibited notable alterations in the peripapillary sclera, including a partial loss of circumferential collagen alignment and a redistribution of the normally observed regional pattern of collagen anisotropic proportion. Linear mixed-model analysis indicated that the mean fiber angle deviation from the circumferential orientation in the peripapillary sclera of highly myopic eyes (23.9° ± 18.2) was statistically significantly higher than that of controls (17.9° ± 12.0; p<0.05). Conclusions: Bulk alterations in the normal posterior scleral collagen microstructure occur in human eyes with high myopia. These changes could reflect remodeling of the posterior sclera during axial lengthening and/or a mechanical adaption to tissue stresses induced by fluid pressure or eye movements that may be exacerbated in enlarged eyes.

Deep tissue analysis of distal aqueous drainage structures and contractile features.

Outflow resistance in the aqueous drainage tract distal to trabecular meshwork is potentially an important determinant of intraocular pressure and success of trabecular bypass glaucoma surgeries. It is unclear how distal resistance is modulated. We sought to establish: (a) multimodal 2-photon deep tissue imaging and 3-dimensional analysis of the distal aqueous drainage tract (DT) in transgenic mice in vivo and ex vivo; (b) criteria for distinguishing the DT from blood and lymphatic vessels; and (c) presence of a DT wall organization capable of contractility. DT lumen appeared as scleral collagen second harmonic generation signal voids that could be traced back to Schlemm's canal. DT endothelium was Prox1-positive, CD31-positive and LYVE-1-negative, bearing a different molecular signature from blood and true lymphatic vessels. DT walls showed prominent filamentous actin (F-actin) labeling reflecting cells in a contracted state. F-actin co-localized with mesenchymal smooth muscle epitopes of alpha-smooth muscle actin, caldesmon and calponin, which localized adjacent and external to the endothelium. Our findings support a DT wall organization resembling that of blood vessels. This reflects a capacity to contract and support dynamic alteration of DT caliber and resistance analogous to the role of blood vessel tone in regulating blood flow.