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.

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.

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.

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.

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.

Iontophoresis-assisted accelerated riboflavin/ultraviolet A scleral cross-linking: A potential treatment for pathologic myopia.

Scleral collagen cross-linking is one of the most promising treatments to control the pathologic process of myopia. However, the exact procedure and its impact on animal models of myopia are still to be explored. We modified the scleral riboflavin/ultraviolet A (UVA) cross-linking procedure with an iontophoresis-assisted drug delivery system and an accelerated UVA irradiation (10 mW/cm2, 9 min) and applied this treatment to an animal model of myopia. Ninety-six New Zealand White rabbits developed relatively stable myopia by visual deprivation and then underwent the modified scleral cross-linking surgery. All the statistics and sample collection were obtained from 4 postoperative time points (1-day, 10-day, 1-month and 3-month groups). We found that the ultimate stress, Young's modulus and physiological Young's modulus of treated myopia sclera were significantly increased and maintained in 4 groups. The abnormal elongation of the myopic eye was effectively controlled 1 month after the treatment and even almost halted 3 months after the treatment. The histochemical assay revealed no notable post-surgery damage or apoptosis in the retina and choroid. Vigorous collagen synthesis was observed in scleral fibroblasts of the treated samples but were rarely observed in the untreated ones under electron microscopy. Furthermore, the remarkable difference in collagen gene expression and protein content between treated and untreated samples also indicated that an alteration in collagen metabolism may be triggered by the treatment. The effectiveness and safety exploration suggested that the modified scleral cross-linking procedure may be a potential method to control the pathologic process of myopia.

Novel characterization and live imaging of Schlemm's canal expressing Prox-1.

Schlemm's canal is an important structure of the conventional aqueous humor outflow pathway and is critically involved in regulating the intraocular pressure. In this study, we report a novel finding that prospero homeobox protein 1 (Prox-1), the master control gene for lymphatic development, is expressed in Schlemm's canal. Moreover, we provide a novel in vivo method of visualizing Schlemm's canal using a transgenic mouse model of Prox-1-green fluorescent protein (GFP). The anatomical location of Prox-1⁺ Schlemm's canal was further confirmed by in vivo gonioscopic examination and ex vivo immunohistochemical analysis. Additionally, we show that the Schlemm's canal is distinguishable from typical lymphatic vessels by lack of lymphatic vessel endothelial hyaluronan receptor (LYVE-1) expression and absence of apparent sprouting reaction when inflammatory lymphangiogenesis occurred in the cornea. Taken together, our findings offer new insights into Schlemm's canal and provide a new experimental model for live imaging of this critical structure to help further our understanding of the aqueous humor outflow. This may lead to new avenues toward the development of novel therapeutic intervention for relevant diseases, most notably glaucoma.

Cholesterol-poly(ethylene) glycol nanocarriers for the transscleral delivery of sirolimus.

The aim of this study was to prepare and characterize cholesterol-poly(ethylene) glycol (chol-PEG) nanocarriers of two different molecular weights (1 and 5 kDa) and to determine their effect on the transscleral retention and permeation of a lipophilic multi-therapeutic agent, sirolimus (rapamycin), with potential application in angiogenic and immunogenic ocular diseases. Sirolimus-containing nanocarriers were prepared using the thin-film hydration method and characterized for their physicochemical properties including size, drug entrapment (EE) and loading (DL) efficiencies, stability, surface charge, morphology, critical micelle concentration (CMC) and thermal properties. Ussing chambers were used to determine the retention and permeability of sirolimus-containing nanocarriers in porcine sclera followed by ultrastructural tissue examination. Sirolimus-containing nanocarriers had an average size of 11.7 nm (chol-PEG 1 kDa) and 13.8 nm (chol-PEG 5 kDa) and zeta potentials of 0.41 and -1.05, respectively. Both nanocarriers had similar transscleral permeabilities (chol-PEG 1 kDa 6.44 × 10(-7) and 5 kDa 6.16 × 10(-7) cm2 s(-1)), and very high scleral retention compared with a free solution of sirolimus (chol-PEG 1 kDa 16.9 µg/g; chol-PEG 5 kDa 7.48 µg/g; free sirolimus 0.57 µg/g). The DL (EE) for chol-PEG 1 and 5 kDa were 2.93% (77.4%) and 3.10% (81.6%), respectively. The CMC values for the nanocarriers were similar to those previously reported in literature (3.85 × 10(-7) M for chol-PEG 1 kDa; 4.26 × 10(-7) M for chol-PEG 5 kDa). In conclusion, chol-PEG nanocarriers successfully loaded sirolimus and resulted in scleral permeation and high retention, which shows potential utility for the topical delivery of lipophilic ocular drugs.

Microstructural changes in sclera under thermo-mechanical effect of 1.56 µm laser radiation increasing transscleral humor outflow.

BACKGROUND AND OBJECTIVES: Pores in the sclera are a candidate pathway for aqueous transport and therefore can be utilized to decrease the intraocular pressure (IOP) in glaucomatous eyes. Since pore formation is a well-known mechanism for stress relaxation in solids, laser-induced creation of pores in cartilage increases hydraulic permeability and promotes tissue regeneration. The aim of this paper is to demonstrate the thermo-mechanical effect of non-destructive laser irradiation on microstructural changes in sclera, in particular pore formation, resulting in substantial increase of water permeability of eye tissues that can be a novel approach to normalize the IOP. MATERIALS AND METHODS: Experiments were performed ex vivo on eight eyes of four mini-pigs and in vivo on eight eyes of four rabbits using pulse repetitive laser radiation of 1.56 µm in wavelength. Twenty laser spots of 0.6 mm in diameter with laser settings (power 0.9 W, pulse duration of 200 milliseconds, pulse repetition rate of 2 Hz) resulting in substantial increase of sclera hydraulic permeability were applied on the sclera at 1-2 mm from the eye limb. Sclera and underlying structures (choroid and ciliary body) of the rabbits' eyes were examined histologically in 1 and 45 days after laser irradiation, atomic force microscope (AFM) was applied before and after laser irradiation. RESULTS: Histological and AFM examinations have clearly recognized laser-assisted stable structural alterations: rarefication of the collagen structure in the laser irradiated zone and formation of sub-micron pores. Laser-induced alterations in the structure of ciliary bodies were small in size and mainly reversible. We have proposed a possible mechanism of the arising pores stabilization due to formation of small stable gas bubbles in sclera tissue. CONCLUSIONS: It is shown, for the first time, that thermo-mechanical effect of pulse repetitive laser irradiation results in pores formation in sclera. That can be a basis of a novel, safe, and effective technique for IOP normalization due to enhancing of uveoscleral outflow under non-destructive laser irradiation of the sclera.

Molecular and chemical investigations and comparisons of biomaterials for ocular surface regeneration.

This study investigated and compared the ultrastructural and chemical properties of representative biomaterials for ocular surface regeneration: a human amniotic membrane (AM) in a basal plate, a human AM in reflected chorion, a preserved AM, and a human corneo-scleral tissue. Assessments of the morphological differences in the extracellular matrices were evaluated by hematoxylin-eosin, Masson's trichrome (for total collagen), and picrosirius-red (for newly synthesized collagen) staining. Assessments of the changes in the molecular structures and chemical compositions of the biomaterials for ocular surface regeneration were evaluated by Raman spectroscopy. A placental AM (52 %) was a dense and thick collagenous structure compared to a reflected AM (23 %). The spectroscopy did not obtain any structural information for a preserved AM. The cornea group (100 %, control) and sclera group (104 %) showed the collagen lamellae and interfibrillar spacing, and a slight inflammatory reaction with more fibrous and granulomatous tissues. There was a formation of newly synthesized collagen in a placental AM, while there were few collagen components in a reflected AM. Human AM tissues showed consistent Raman spectra and the characteristic collagen bands, similar to the corneal and scleral tissues. Therefore, these findings suggest that human placental AM and reflected AM are structurally suitable for scleral and corneal surface regeneration, respectively, while human placental or preserved AM and reflected AM are molecularly and chemically suitable for corneal and scleral surface regeneration, respectively.

Primitive tumor of lamina fusca: original findings

In a meeting of the Pan-American Association of Ophthalmic Pathology, held in Los Angeles, CA, on October 10, 1991, at the Doheny Eye Institute, C.O. Degrazia proposed the name LOFONEUROGONIOMA for a single undifferentiated intraocular tumor originated in the cells of lamina fusca, with undifferentiated cells of expressive differentiation into Schwann cell, melanoblast and neuroendocrine cell lineages. In view of trimorphism, a pathologist may be led to a diagnosis of malignant melanoma, schwannoma or endocrine cell tumor. After this date, an article was published in Revista da AMRIGS, Porto Alegre, 52 (4): 261-272, oct-dec 2008, with collaboration of experts from the fields of Immunohistochemistry and electron microscopy. The current publication is the third one and its main purpose is to highlight the original characteristics of such a tumor.

Na reunião da Associação Pan-americana de Patologia Oftálmica, realizada em Los Angeles CA, 10 de Outubro de 1991, no DOHENY EYE INSTITUTE, C.O. Degrazia propôs o nome de LOFONEUROGONIOMA para um tumor solitário intraocular, indiferenciado, originado nas células da lâmina fusca, com células indiferenciadas de expressiva diferenciação para a linhagem schwannoblástica, melanoblástica e neuroendócrina. Em face do trimorfismo, o patologista pode ser conduzido para os diagnósticos de melanoma, schwannoma malignos, ou tumor de células endócrinas. Foi feita após essa data uma publicação na Revista da AMRIGS. Porto Alegre, 52 (4): 261-272, out.-dez 2008, na qual consta a lista dos colaboradores. A publicação atual é a terceira, tendo como finalidade principal colocar em relevo as originalidades de semelhante tumor.

Intrascleral outflow after deep sclerectomy with absorbable and non-absorbable implants in the rabbit eye.

BACKGROUND: The purpose of the study is an analysis of intrascleral drainage vessels formed in rabbits' eyes after non-penetrating deep sclerectomy (NPDS) with absorbable and non-absorbable implants, and comparison to eyes in which surgery was performed without implanted material. MATERIAL/METHODS: NPDS was carried out in 12 rabbits, with implantation of non-absorbable methacrylic hydrogel (N=10 eyes) or absorbable cross-linked sodium hyaluronate (N=6 eyes), or without any implant (N=8 eyes). All the animals were euthanized 1 year after surgery. Twenty-one eyeballs were prepared for light microscopy and 3 were prepared for transmission electron microscope (TEM) analysis. Aqueous humour pathways were stained with ferritin in 6 eyeballs. RESULTS: By light microscopy, small vessels adjacent to the areas of scarring were the most common abnormality. Vessel density was significantly higher in operated sclera compared to normal, healthy tissue, regardless of the type of implant used. The average vessel densities were 2.18±1.48 vessels/mm2 in non-implanted sclera, 2.34±1.69 vessels/mm2 in eyes with absorbable implants, and 3.64±1.78 vessels/mm2 in eyes with non-absorbable implants. Analysis of iron distribution in ferritin-injected eyes showed a positive reaction inside new aqueous draining vessels in all groups. TEM analysis showed that the ultrastructure of new vessels matched the features of the small veins. CONCLUSIONS: Aqueous outflow after NPDS can be achieved through the newly formed network of small intrascleral veins. Use of non-absorbable implants significantly increases vessel density in the sclera adjacent to implanted material compared to eyes in which absorbable implants or no implants were used.

Quantitative measurement of wound architecture in microincision cataract surgery.

PURPOSE: To evaluate whether changes in internal wound architecture after phacoemulsification can be measured quantitatively by analysis of scanning electron micrographs. SETTING: Baylor College of Medicine, Houston, Texas, USA. DESIGN: Experimental study. METHODS: Two comparative studies in human cadaver eyes were performed using coaxial small-incision cataract surgery (SICS), bimanual microincision cataract surgery (MICS), coaxial MICS, and a variety of phacoemulsification tips (MicroSurgical Technologies, Microphaco Tapered, Microtip Turbosonics, and Microphaco Mackool). After surgery, the cornea and scleral rims were harvested and digital scanning electron micrographs were taken. The internal corneal wound was analyzed using measurement software to determine the area of endothelial cell loss and length of Descemet membrane tears. RESULTS: Quantifiable differences were observed between combinations of techniques and tip designs. The mean area of endothelial cell loss was 2.93 ± 0.31 mm(2) (SD) after coaxial MICS (n = 4) and 2.85 ± 0.54 mm(2) after bimanual MICS (n = 4). However, after normalizing for differences in tip or sleeve diameter, the area of endothelial cell loss ranged from 1.4-fold to 1.7-fold less with coaxial SICS than with bimanual MICS with Microphaco Tapered, Microtip Turbosonics, and Microphaco Mackool tips. The mean total length of tearing was 1.38 ± 0.38 mm for bimanual MICS (n = 4) and 0.84 ± 0.61 mm for coaxial MICS (n = 4). CONCLUSION: The length and area of corneal wounds could be quantitated with accuracy and the measurements could be used to make quantifiable comparisons of phacoemulsification techniques and tips. FINANCIAL DISCLOSURE: Dr. Dimalanta is an employee of Alcon Research, Ltd. No other author has a financial or proprietary interest in any material or method mentioned.

Effects of 7-methylxanthine on the sclera in form deprivation myopia in guinea pigs.

PURPOSE: The aim of this study was to determine the effect of the adenosine receptor antagonist 7-methylxanthine (7-MX) on form deprivation myopia in 3-week-old guinea pigs. METHODS: Two groups of 3-week-old guinea pigs were subjected to monocular deprivation (MD) using a diffuser and fed either 7-MX (300 mg/kg body weight; n = 7) or vehicle control (saline at an equal volume to 7-MX; n = 7). A control group (n = 6) was not subjected to form deprivation. Ocular refraction, axial length and body weight were measured at the start and after 21 days. The thickness of the posterior sclera was measured by light microscopy and the collagen fibril diameter in the inner, middle and outer layers of the sclera was measured by electron microscopy. RESULTS: In the vehicle control group, 21 days of MD produced significant amounts of myopia, axial elongation, thinning of the posterior sclera and thinning of the median collagen fibril diameter in the posterior sclera relative to the contralateral eyes. In the guinea pigs fed with 7-MX, however, form deprivation produced significantly less myopia and axial elongation compared with vehicle control animals. The 7-MX-treated animals exhibited a thickening of the posterior sclera in both the MD eye and the contralateral eye. In the 7-MX-treated animals, the median collagen fibril diameter in the posterior sclera was not reduced by form deprivation. CONCLUSIONS: Treatment with 7-MX appears to not only decrease the amount of myopia by around 50% and eliminate the eye elongation induced by form deprivation in guinea pigs, but also to prevent form deprivation myopia-related scleral changes, such as thinning of the sclera and thinning of the collagen fibril diameter in the posterior sclera.

Irreversible optical clearing of sclera by dehydration and cross-linking.

This study manipulates both clear cornea and opaque sclera by two dehydration processes for revealing the relationship between altered tissue structures and change in optical functions. In contrast to the high levels of light scattering in dehydrated tissues by critical point dry, a simple dehydration at 4-8 °C effectively and significantly improved their visible-light transmission, even in the sclera, with accompanying dense fiber packing. Further improvement in visible-light transmission, from 40-50% to 80-90%, has been achieved by flatting tissue surface with cover glasses during dehydration at low temperature. Such optical clearing of sclera by dehydration is reversible. However, chemical cross-linking effectively stabilizes their densely packed microscopic structures and visible-light transmission at over 50% irreversibly, even at wet conditions. Interestingly, the repetition of both low temperature dehydration/cross-linking treatments effectively reduced the required amounts of cross-linking reagents to keep a high transparency. Wet transparent cross-linked sclera can also show a characteristic strong tensile strength. Furthermore, rabbit corneal epithelium has regenerated on the transparent sclera with cross-linking in vitro.

Genetic deletion of the adenosine A2A receptor confers postnatal development of relative myopia in mice.

PURPOSE: To critically evaluate whether the adenosine A2A receptor (A2AR) plays a role in postnatal refractive development in mice. METHODS: Custom-built biometric systems specifically designed for mice were used to assess the development of relative myopia by examining refraction and biometrics in A2AR knockout (KO) mice and wild-type (WT) littermates between postnatal days (P)28 and P56. Ocular dimensions were measured by customized optical coherence tomography (OCT), refractive state by eccentric infrared photorefraction (EIR), and corneal radius of curvature by modified keratometry. Scleral collagen diameter and density were examined by electron microscopy on P35. The effect of A2AR activation on collagen mRNA expression and on soluble collagen production was examined in cultured human scleral fibroblasts by real-time RT-PCR and a collagen assay kit. RESULTS: Compared with WT littermates, the A2AR KO mice displayed relative myopia (average difference, 5.1 D between P28 and P35) and associated increases in VC depth and axial length from P28 to P56. Furthermore, the myopic shift in A2AR KO mice was associated with ultrastructural changes in the sclera: Electron microscopy revealed denser collagen fibrils with reduced diameter in A2AR KO compared with WT. Last, A2AR activation induced expression of mRNAs for collagens I, III, and V and increased production of soluble collagen in cultured human scleral fibroblasts. CONCLUSIONS: Genetic deletion of the A2AR promotes development of relative myopia with increased axial length and altered scleral collagen fiber structure during postnatal development in mice. Thus, the A2AR may be important in normal refractive development.

Two-photon microscopy of deep intravital tissues and its merits in clinical research.

Multiphoton excitation laser scanning microscopy, relying on the simultaneous absorption of two or more photons by a molecule, is one of the most exciting recent developments in biomedical imaging. Thanks to its superior imaging capability of deeper tissue penetration and efficient light detection, this system becomes more and more an inspiring tool for intravital bulk tissue imaging. Two-photon excitation microscopy including 2-photon fluorescence and second harmonic generated signal microscopy is the most common multiphoton microscopic application. In the present review we take diverse ocular tissues as intravital samples to demonstrate the advantages of this approach. Experiments with registration of intracellular 2-photon fluorescence and extracellular collagen second harmonic generated signal microscopy in native ocular tissues are focused. Data show that the in-tandem combination of 2-photon fluorescence and second harmonic generated signal microscopy as two-modality microscopy allows for in situ co-localization imaging of various microstructural components in the whole-mount deep intravital tissues. New applications and recent developments of this high technology in clinical studies such as 2-photon-controlled drug release, in vivo drug screening and administration in skin and kidney, as well as its uses in tumourous tissues such as melanoma and glioma, in diseased lung, brain and heart are additionally reviewed. Intrinsic emission two-modal 2-photon microscopy/tomography, acting as an efficient and sensitive non-injurious imaging approach featured by high contrast and subcellular spatial resolution, has been proved to be a promising tool for intravital deep tissue imaging and clinical studies. Given the level of its performance, we believe that the non-linear optical imaging technique has tremendous potentials to find more applications in biomedical fundamental and clinical research in the near future.

Acanthamoeba sclerokeratitis.

BACKGROUND: Acanthamoeba scleritis is an uncommon but severe complication of acanthamoeba keratitis. We report the clinical and histopathologic features of a patient with acanthamoeba sclerokeratitis. METHODS: Review of the patient's clinical records and histopathologic examination of the globe including light microscopy and transmission electron microscopy. RESULTS: Review of the clinical record of the patient revealed a past ocular history of penetrating keratoplasty for persistent acanthamoeba keratitis. Later in the course of treatment, the patient developed nodular necrotizing scleritis with culture-proven viable acanthamoeba in this area. She underwent enucleation of the eye for persistent acanthamoeba sclerokeratitis. Histopathologic examination of the globe revealed no acanthamoeba cysts or trophozoites at the site of crotherapy. CONCLUSION: Our study provides evidence for the invasion of acanthamoeba organisms into the sclera in a case of acanthamoeba keratitis. The presence of trophozites in scleral tissue may exacerbate the immune response leading to nodular scleritis.

Fine structure of the interface between the anterior limiting lamina and the anterior stromal fibrils of the human cornea.

PURPOSE: To use transmission electron microscopy (TEM) to investigate further the ultrastructural details of the collagen fibrils linking the anterior limiting lamina (ALL; Bowman's membrane) of the human cornea to the anterior stromal lamellae. METHODS: Six disease-free corneas from donors aged 42 to 82 years were fixed (2% glutaraldehyde in 80 mM sodium cacodylate) and processed for TEM within 72 hours postmortem. A series of overlapping images, at 10,204x magnification, of the central corneal ALL-stroma interface were assembled. The features of the terminal ends of fibril bundles at the interface with the anterior stroma were quantitatively assessed. RESULTS: TEM revealed apparently terminating anterior stromal fibril bundles adjacent to the ALL. These terminating lamellae (7.8 per 100 mum) were embedded in an electron-dense material within the surrounding stromal matrix and were termed electron-dense formations (EDFs). The mean width of these stromal features was 1.6 mum. At intervals, anterior stromal lamellae approached the ALL and, in a shallow manner, inserted into the ALL. Such projections (5.4 per 100 mum) into the ALL were, on average, less than 1 mum. Numerous fibrils (29.8 per 100 mum) extended from the ALL into the stroma with a mean length of 0.8 mum. CONCLUSIONS: The interface the ALL forms with the anterior stroma is complex, and TEM revealed at least three different types of fibrillar arrangements, which may serve optical requirements rather than provide a structural function.