Temperature effects on the disappearance and reappearance of corneal-endothelium primary cilia.

INTRODUCTION: To elucidate the specific functions of the primary cilia in corneal endothelial cells (CECs) by investigating the histological changes of corneal endothelium exposed at low temperature. STUDY DESIGN: Experimental study. METHODS: This study involved corneas freshly obtained from Japanese white rabbits preserved in Optisol™-GS (Bausch & Lomb) corneal storage medium at 4 °C for 0, 1, and 7 days. Corneas preserved for 7 days were also incubated at 37 °C in culture media for an additional 2 days. A rabbit CEC line was also preserved in Optisol™-GS at 4 °C for 0 and 1 day. The corneal endothelium specimens and CECs were then assessed by immunostaining and scanning electron-microscopy (SEM). RESULTS: Immediately post isolation, the CECs of the specimens showed positive immunostaining for primary cilia (i.e., approximately 20%) via anti-acetylated alpha Tubulin antibody and SEM observation. Primary cilia were found to have attenuated/disappeared on the corneal endothelium specimens preserved for 1 or 7 days at 4 °C. After an additional 2-day incubation at 37 °C, primary cilia reappeared on the corneal endothelium specimens (approximately 20%). The disappearance of cilia during the preservation period was also observed in the immortalized CECs. CONCLUSION: The findings in this study using rabbit corneas indicate that the primary cilia of corneal endothelium preserved at low temperature disappeared, then reappeared after returning to body temperature, suggesting that temperature has a direct effect on the primary cilia of corneal endothelium.

Potential participation of CTRP6, a complement regulator, in the pathology of age related macular degeneration.

PURPOSE: To investigate the localized expression of C1q/tumor necrosis factor related protein (CTRP) 6 in human age-related macular degeneration (AMD) retinal tissues. EXPERIMENTAL STUDY DESIGN: 4 AMD and 3 non-AMD whole eyes of Caucasian donors were used. Eyecups were excised at Eye Bank CorneaGen, Inc. METHODS: To elucidate the effects of CTRP6, C3b was measured by an enzyme-linked immunosorbent-like assay. CFB versus CTRP6 competitive binding assay was applied to clarify the inhibition by CTRP6 of C3bBb complex formation. The cornea, iris, lens, and vitreous were removed and the eyes were cut into a posterior eye-cup including the retina, choroid, and sclera. Six-µm-thick serial sections of frozen samples underwent hematoxylin-eosin (HE) staining and indirect immunohistochemical staining using primary antibodies, anti-CTRP6, -CTRP5, -CTRP10, -Complement factor H (CFH) and -Clusterin (CLU). Results The two in vitro studies confirmed that CTRP6 has an inhibitory effect on alternative pathways of complement (APC) function and that the molecular target of CTRP6 is the inhibition of the formation of C3bBb. Localized expression for CTRP6 and CFH was found in the drusen of the AMD eyes, both associated with APC inhibition, CLU associated with membrane-attack complex (MAC) inhibition, and CTRP5 associated with retinal degeneration. CONCLUSION: The localized expression of CTRP6 in the drusen of AMD eyes may open a new insight into the possible involvement of APC regulatory factors in the pathogenesis of AMD, together with the known CFH so far analyzed solely as an APC inhibitor.

Chemical proteasome inhibition as a novel animal model of inner retinal degeneration in rats.

Chemical proteasome inhibition has been a valuable animal model of neurodegeneration to uncover roles for the ubiquitin-proteasome system in the central nervous system. However, little is known about the effects of chemical proteasome inhibitors on retinal integrity. Therefore, we characterized the effects of structurally different chemical proteasome inhibitors on the retinal morphology and the mechanisms of their action in the normal adult rat eyes. Intravitreal injection of MG-262 and other proteasome inhibitors led to inner retinal degeneration. MG-262-induced inner retinal degeneration was accompanied by reduced proteasome activity, increased poly-ubiquitinated protein levels, and increased positive immunostaining of ubiquitin, 20S proteasome subunit and GADD153/CHOP in the retina. Its retinal degenerative effect was also associated with reduced retinal neurofilament light chain gene expression, reflecting retinal ganglion cell death. MG-262-induced neurofilament light chain downregulation was largely resistant to pharmacological modulation including endoplasmic reticulum stress, apoptosis or MAP kinase inhibitors. Thus, this study provides further evidence of roles for the ubiquitin-proteasome system in the maintenance of the retinal structural integrity. Chemical proteasome inhibition may be used as a novel animal model of inner retinal degeneration, including retinal ganglion cell loss, which warrants further analysis of the molecular mechanisms underlying its retinal degenerative effect.

Biopsy of recurrent nasolacrimal duct obstruction using sheath-guided dacryoendoscopy.

PURPOSE: The purpose of this article is to present a novel technique, as well the histopathological findings, of dacryoendoscopic guided nasolacrimal duct (NLD) biopsy for recurrent nasolacrimal duct obstruction (NLDO). METHODS: This study involved subjects with recurrent NLDO. Direct endoscopic probing or sheath-guided endoscopic probing was used for the initial intubation in all treated eyes, and the stent had been removed at between 2 and 11 months (mean 3.5 months) post-intubation with dacryoendoscopic confirmation of patency and mucosal regeneration. Biopsy specimens were obtained by scraping the recurrent lesion by sheath advancement. Histopathological examination and immunohistochemical (IHC) staining were performed. RESULTS: In five patients (two males and three females, mean age: 71.2 ± 5.6 years [range: 61-78 years]) with recurrent NLDO, biopsy specimens were obtained from six ducts of six eyes, and stratified epithelium and a mixed inflammatory cell infiltrates were identified. IHC staining was positive for cytokeratin (CK)4 and CK13, and negative for paired box protein Pax-6. CONCLUSIONS: This novel technique enabled a minimally invasive biopsy of the NLD to be obtained, and IHC staining indicated the presence of mucus epithelium, thus suggesting squamous metaplasia of the usual respiratory epithelium which likely occurs secondary to chronic inflammation.

A new dry eye mouse model produced by exorbital and intraorbital lacrimal gland excision.

Chronic dry eye is an increasingly prevalent condition worldwide, with resulting loss of visual function and quality of life. Relevant, repeatable, and stable animal models of dry eye are still needed. We have developed an improved surgical mouse model for dry eye based on severe aqueous fluid deficiency, by excising both the exorbital and intraorbital lacrimal glands (ELG and ILG, respectively) of mice. After ELG plus ILG excision, dry eye symptoms were evaluated using fluorescein infiltration observation, tear production measurement, and histological evaluation of ocular surface. Tear production in the model mice was significantly decreased compared with the controls. The corneal fluorescein infiltration score of the model mice was also significantly increased compared with the controls. Histological examination revealed significant severe inflammatory changes in the cornea, conjunctiva or meibomian glands of the model mice after surgery. In the observation of LysM-eGFP(+/-) mice tissues, postsurgical infiltration of green fluorescent neutrophils was observed in the ocular surface tissues. We theorize that the inflammatory changes on the ocular surface of this model were induced secondarily by persistent severe tear reduction. The mouse model will be useful for investigations of both pathophysiology as well as new therapies for tear-volume-reduction type dry eye.

Spermidine Oxidation-Mediated Degeneration of Retinal Pigment Epithelium in Rats.

Retinal pigment epithelium (RPE) degeneration is a crucial event in dry age-related macular degeneration and gyrate atrophy. The polyamine spermidine has been shown to induce RPE cell death in vitro. The present study aimed to establish a novel in vivo model of spermidine-induced RPE degeneration and to determine whether spermidine-induced RPE cell death involves oxidative mechanisms. In this study, spermidine caused ARPE-19 cell death in a concentration-dependent manner. This effect was prevented by removal of serum from the culture medium or treatment with amine oxidase inhibitors, N-acetylcysteine (NAC), or aldehyde dehydrogenase (ALDH). Intravitreal injection of spermidine into rats significantly increased the permeability of the blood-retinal barrier and decreased the amplitudes of scotopic electroretinogram a- and b-waves. Histological analysis revealed that spermidine induced vacuolation, atrophy, and dropout of RPE cells, leading to the disruption of photoreceptor outer segments. Simultaneous intravitreal administration of NAC and ALDH with spermidine prominently inhibited the functional and morphological changes induced by spermidine. In conclusion, this study demonstrated that the intravitreal administration of spermidine induced RPE cell dysfunction and death followed by photoreceptor degeneration in rats. These effects of spermidine are thought to be mediated by oxidative stress and a toxic aldehyde generated during spermidine oxidation.

Allogeneic Sensitization and Tolerance Induction After Corneal Endothelial Cell Transplantation in Mice.

PURPOSE: We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayed-type hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance. METHOD: The corneal eyecups from C57BL/6 mice were prepared. The epithelial layer was detached with EDTA solution and treated with trypsin to release mouse-derived primary corneal endothelial cells (mpCECs). The mpCECs (1 × 104 cells) were transplanted into the AC of the eye or subcutaneously (SC) into the neck of BALB/c mice. In the mouse model of endothelial cell transplantation, the endothelial cells in a 2-mm central area of the cornea were eliminated by cryoinjury. The mpCEC transplant model was evaluated by measuring allogeneic cell survival and corneal thickness. The allospecific DTH response and ACAID induction were evaluated 1 week after transplantation. The long-term transplantation tolerance was evaluated by observing a secondary penetrating keratoplasty (PKP) performed on the same donor C57BL/6 mice. RESULTS: The SC injection of mpCECs induced a DTH response, whereas the AC injection induced ACAID. However, eyes inflamed by cryoinjury showed neither the DTH response nor ACAID following AC injection. The mpCECs survived for at least 1 week after injection. Penetrating keratoplasty allografts at 8 weeks after mpCEC transplantation survived indefinitely (100%). CONCLUSIONS: The mpCECs display low allogenicity in the AC and are capable of inducing allogeneic tolerance. Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation.

Intravital imaging of the cellular dynamics of LysM-positive cells in a murine corneal suture model.

BACKGROUND: Corneal suturing is a surgical procedure used in patients with corneal trauma or transplants. It was reported that endogenous neutrophils are brightly labelled in gene-targeted mice expressing enhanced green fluorescent protein (eGFP) under the control of the endogenous lysozyme M promoter (LysM-eGFP mice). METHODS: We applied intravital imaging methods to analyse in vivo the dynamics of LysM-positive granulocytes (neutrophils) in LysM-eGFP mice with corneal sutures and examined their role in the elicitation of neutrophil infiltration. RESULTS: We found that in the presuturing state, neutrophils strongly positive for LysM were located in the periphery of the corneal stromal layer; none were present in the centre of the cornea. After introducing a corneal suture, neutrophils accumulated in limbal vessels and then migrated to the corneal side and the conjunctival side, suggesting that they derived from limbal vessels. Thereafter they accumulated towards the central corneal area, arriving at the suture about 7 h after its placement. Although corneal sutures may elicit the continuous infiltration of neutrophils, their number was markedly decreased by day 1 after suture removal and continued to decrease thereafter. CONCLUSIONS: Our results showed that corneal sutures may elicit the continuous infiltration of neutrophils.

Testosterone stimulates glucose uptake and GLUT4 translocation through LKB1/AMPK signaling in 3T3-L1 adipocytes.

Decreases in serum testosterone concentrations in aging men are associated with metabolic disorders. Testosterone has been reported to increase GLUT4-dependent glucose uptake in skeletal muscle cells and cardiomyocytes. However, studies on glucose uptake occurring in response to testosterone stimulation in adipocytes are currently not available. This study was designed to determine the effects of testosterone on glucose uptake in adipocytes. Glucose uptake was assessed with 2-[(3)H] deoxyglucose in 3T3-L1 adipocytes. GLUT4 translocation was evaluated in plasma membrane (PM) sheets and PM fractions by immunofluorescence and immunoblotting, respectively. Activation of GLUT4 translocation-related protein kinases, including Akt, AMPK, LKB1, CaMKI, CaMKII, and Cbl was followed by immunoblotting. Expression levels of androgen receptor (AR) mRNA and AR translocation to the PM were assessed by real-time RT-PCR and immunoblotting, respectively. The results showed that both high-dose (100 nM) testosterone and testosterone-BSA increased glucose uptake and GLUT4 translocation to the PM, independently of the intracellular AR. Testosterone and testosterone-BSA stimulated the phosphorylation of AMPK, LKB1, and CaMKII. The knockdown of LKB1 by siRNA attenuated testosterone- and testosterone-BSA-stimulated AMPK phosphorylation and glucose uptake. These results indicate that high-dose testosterone and testosterone-BSA increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes by inducing the LKB1/AMPK signaling pathway.

Mesenchymal-epithelial cell interactions and proteoglycan matrix composition in the presumptive stem cell niche of the rabbit corneal limbus.

PURPOSE: To investigate whether mesenchymal-epithelial cell interactions, similar to those described in the limbal stem cell niche in transplant-expired human eye bank corneas, exist in freshly enucleated rabbit eyes and to identify matrix molecules in the anterior limbal stroma that might have the potential to help maintain the stem cell niche. METHODS: Fresh limbal corneal tissue from adult Japanese white rabbits was obtained and examined in semithin resin sections with light microscopy, in ultrathin sections with transmission electron microscopy, and in three-dimensional (3D) reconstructions from data sets of up to 1,000 serial images from serial block face scanning electron microscopy. Immunofluorescence microscopy with five monoclonal antibodies was used to detect specific sulfation motifs on chondroitin sulfate glycosaminoglycans, previously identified in association with progenitor cells and their matrix in cartilage tissue. RESULTS: In the rabbit limbal cornea, while no palisades of Vogt were present, the basal epithelial cells stained differentially with Toluidine blue, and extended lobed protrusions proximally into the stoma, which were associated with interruptions of the basal lamina. Elongate processes of the mesenchymal cells in the superficial vascularized stroma formed direct contact with the basal lamina and basal epithelial cells. From a panel of antibodies that recognize native, sulfated chondroitin sulfate structures, one (6-C-3) gave a positive signal restricted to the region of the mesenchymal-epithelial cell associations. CONCLUSIONS: This study showed interactions between basal epithelial cells and subjacent mesenchymal cells in the rabbit corneal limbus, similar to those that have been observed in the human stem cell niche. A native sulfation epitope in chondroitin sulfate glycosaminoglycans exhibits a distribution specific to the connective tissue matrix of this putative stem/progenitor cell niche.

Establishment of a human corneal epithelial cell line lacking the functional TACSTD2 gene as an in vitro model for gelatinous drop-like dystrophy.

PURPOSE: Gelatinous drop-like corneal dystrophy (GDLD) is characterized by subepithelial amyloid deposition that engenders severe vision loss. The exact mechanism of this disease has yet to be elucidated. No fundamental treatment exists. This study was conducted to establish an immortalized corneal epithelial cell line to be used as a GDLD disease model. METHODS: A corneal tissue specimen was obtained from a GDLD patient during surgery. Corneal epithelial cells were enzymatically separated from the cornea and were dissociated further into single cells. The epithelial cells were immortalized by the lentiviral transduction of the simian virus 40 (SV40) large T antigen and human telomerase reverse transcriptase (hTERT) genes. For the immortalized cells, proliferative kinetics, gene expressions, and functional analyses were performed. RESULTS: The immortalized corneal epithelial cells continued to proliferate despite cumulative population doubling that exceeded 100. The cells showed almost no sign of senescence and displayed strong colony-forming activity. The cells exhibited a low epithelial barrier function as well as decreased expression of tight-junction-related proteins claudin 1 and 7. Using the immortalized corneal epithelial cells derived from a GDLD patient, we tested the possibility of gene therapy. CONCLUSIONS: We established an immortalized corneal epithelial cell line from a GDLD patient. The immortalized cells exhibited cellular phenotypes similar to those of in vivo GDLD. The immortalized cells are thought to be useful for the development of new therapies for treating GDLD corneas and for elucidation of the pathophysiology of GDLD.

Interaction between conjunctival epithelial cells and mast cells induces CCL2 expression and piecemeal degranulation in mast cells.

PURPOSE: Intraepithelial mast cells are observed in giant papillae tissue samples obtained from patients with atopic keratoconjunctivitis (AKC)/vernal keratoconjunctivitis (VKC). We examined the roles of interaction between the conjunctival epithelial cells and mast cells. METHODS: The interaction between human mast cells and conjunctival epithelial cells (HCjE) was investigated using a coculture model. Protein array analysis, ELISA, and real-time PCR were performed to test the interaction. Tissue samples (n = 6) from giant papillae were resected for therapeutic purposes, and subjected to immunohistological analysis of CCL2 expression. Recombinant CCL2 (10 ng/mL) was reacted with the cultured human mast cells and ultrastructural analysis was performed. A ragweed (RW)-induced mouse experimental allergic conjunctivitis model was used to examine ccl2 mRNA expression and mast cell morphology. RESULTS: Protein array and real-time PCR analyses showed that CCL2 protein/mRNA expression was induced by mast cell-HCjE coculture. Upregulation of CCL2 mRNA was observed in mast cells, whereas in situ CCL2 expression was observed at the conjunctival epithelium of the giant papillae by immunohistochemistry. Ultrastructural analysis showed that recombinant CCL2 treatment induced piecemeal degranulation (PMD) in the mast cells. Ultrastructural analysis of tissues from the giant papillae showed PMD of mast cells within the conjunctival epithelial cells. The RW-induced experimental allergic conjunctivitis model showed increased ccl2 mRNA expression and PMD morphology in the conjunctivae. CONCLUSIONS: Mast cell-conjunctival epithelial cell interaction induces CCL2 expression and subsequent PMD.

Immunohistochemical analysis of inflammatory limbal conjunctiva adjacent to Mooren's ulcer.

BACKGROUND/AIMS: To examine the characteristics of infiltrating cells in conjunctival tissues adjacent to the peripheral corneal ulcers of Mooren's ulcer. METHODS: This study involved four eyes of four patients with Mooren's ulcer and who were considered to be in need of surgical treatment. The patients' resected conjunctival tissues were embedded and frozen. The tissue sections were then subjected to H&E and immunohistochemical staining. The stained sections were observed and the characteristics of the infiltrating cells in the conjunctival tissues were pathologically examined. RESULTS: In all patients, infiltration of inflammatory cells was observed in the submucosal connective tissue of the conjunctiva. Immunohistochemical analysis revealed inflammatory cell infiltration into the submucosal layer of the conjunctiva that was mainly composed of CD3-positive and CD45RO-positive cells. Some of these cells also showed positive reactivity with CD4, yet very few cells showed positive reactivity with CD8. In addition, infiltration of the cells indicating CD68 positivity was frequent in a few cases. CONCLUSIONS: In the four Mooren's ulcer cases, infiltrating cells in the submucosa of the conjunctival tissues adjacent to the ulcerative cornea were found to be mainly composed of helper T lymphocytes and macrophages. Our results show that helper T cells and macrophages contribute to the pathogenesis of Mooren's ulcer.

Characterization of monocarboxylate uptake and immunohistochemical demonstration of monocarboxylate transporters in cultured rabbit corneal epithelial cells.

OBJECTIVES: This study aimed to characterize the mechanisms of monocarboxylate uptake by cultured rabbit corneal epithelial cells (RCECs) using l- and d-lactic acids as model substrates. METHODS: l-/d-Lactic acid uptake was evaluated by measuring the accumulation in confluent RCECs. Also, we demonstrated the distribution of monocarboxylate transporters (MCTs) in RCECs by immunohistochemistry. KEY FINDINGS: The accumulation of (14) C-labelled l- and d-lactic acids was dependent on time, pH and temperature. The Arrhenius plots of the uptake were biphasic. The initial uptake of (14) C-labelled l-lactic acid exhibited concentration dependence and was greater than that of the d-isomer. The initial uptake of (14) C-labelled l- and d-lactic acids involved saturable and nonsaturable processes; the saturable process exhibited higher affinity for l-lactic acid than for the d-isomer. l-/d-lactic acid uptake was inhibited by chiral monocarboxylate in a stereoselective manner. The uptake of (14) C-labelled l- and d-lactic acids was sensitive to metabolic inhibitors and other monocarboxylates. MCT expression in RCECs was confirmed immunohistochemically. In particular, MCT2 expression was detected in RCECs, whereas MCT1, MCT4 and MCT5 expression was detected in the surface layer. CONCLUSION: These results indicate that the carrier-mediated transport system specific for monocarboxylates elicits lactic acid uptake in RCECs. Therefore, the transcorneal permeation of drugs with a monocarboxylic moiety may be dependent on the activity of a specific pH-dependent transporter as well as passive diffusion according to the pH-partition theory.

Simultaneous analysis of multiple cytokines in the vitreous of patients with sarcoid uveitis.

PURPOSE: Levels of some cytokines are significantly higher in the vitreous fluid of patients with acute uveitis than in normal vitreous fluid. The authors sought to determine which proinflammatory cytokines were upregulated in the vitreous fluid of patients with ocular sarcoidosis. METHODS: Samples of vitreous fluid were collected from patients with sarcoid uveitis and from nonsarcoid control patients with idiopathic epiretinal membrane. The levels of 27 proinflammatory cytokines were measured with a multiplex beads array system. Postvitrectomy macular thickness was also measured by using spectral domain optical coherence tomography (SD-OCT). To assess the relationship between cytokine levels and disease stage, the authors divided patients into three groups based on macular thickness 1 month after operation. RESULTS: The vitreous levels of 17 cytokines were significantly higher in patients with ocular sarcoidosis than in nonsarcoid controls. Serum levels of interferon γ-induced protein 10 (IP-10) were also higher in ocular sarcoidosis patients than in nonsarcoid controls. Conversely, serum levels of interleukin (IL) 15 in ocular sarcoidosis patients were lower than in the control group. Analysis of cytokine levels and macular thickness revealed that IL-1ra, IL-4, IL-8, IFN-γ, IP-10, monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1ß, and regulated on activation, normal T-cell expressed and secreted (RANTES) were significantly upregulated in patients with thin cystoid macular edema group. CONCLUSIONS: Patients with ocular sarcoidosis had elevated levels of proinflammatory cytokines in vitreous fluids. Different cytokines might contribute to different stages of macular edema.

A novel mutation (p.Glu1389AspfsX16) of the phosphoinositide kinase, FYVE finger containing gene found in a Japanese patient with fleck corneal dystrophy.

PURPOSE: The phosphoinositide kinase, FYVE finger containing (PIKFYVE) gene has been identified as a gene responsible for fleck corneal dystrophy (FCD). The purpose of this study is to report a novel mutation of the PIKFYVE gene in a Japanese patient with fleck corneal dystrophy. METHODS: Slit-lamp microscopy, corneal topography, and optical coherence tomography were performed for the clinical examination of the patient's eye. For genetic analysis, peripheral blood was obtained from the patient and her sister. DNA was extracted from the blood and subjected to mutation analysis by sequencing of the PIKFYVE gene. The sequencing results were validated with a PCR-fragment length polymorphism analysis. RESULTS: A 63-year-old woman presented at our clinic with complaints of decreased vision and metamorphopsia in her right eye occurring 1 month before presentation. Both eyes exhibited small, dot-like, white flecks scattered throughout all layers of the corneal stroma, which corresponds to the typical FCD phenotype. The opacities were relatively dominant at the peripheral region of the cornea, yet were found throughout the entire cornea. Sequence analysis revealed that the patient has a heterozygous c.4166_4169delAAGT mutation located at exon 24 of the PIKFYVE gene that may cause p.Glu1389AspfsX16 flame-shift mutation, which has never before been reported for FCD. CONCLUSIONS: To the best of our knowledge, this is the first study to show that a novel mutation (p.Glu1389AspfsX16) causing the truncation of the PIKFYVE protein causes fleck corneal dystrophy in the Japanese population.