Scleral Fibroblasts Research Articles

Effects of Mechanical Stimulation on MMP-2 and TIMP-2 Expression of Scleral Fibroblasts after Posterior Sclera Reinforcement

To understand the effect of mechanical stimulation on posterior sclera reinforcement (PSR), the rabbit scleral fibroblasts after PSR were subjected to stretch in vitro and MMP-2 and TIMP-2 expression of scleral fibroblasts were evaluated. Three-week-old rabbits were monocularly performed by eyelid suturation randomly to prepare experimental myopia eye. After 60 days, the experimental myopia eyes were treated by PSR. After 6 months, the posterior pole scleral fibroblasts (normal sclera - group A, sclera after operation - group B and fusion region of sclera and reinforcing band group C) were isolated and cultured in vitro. The cells were subjected to cyclic stretch regimens (sine wave, 3% and 6% elongation amplitude, 0.1Hz, 48h duration) by FX-4000 Tension System. The MMP-2 and TIMP-2 expression of scleral fibroblasts were evaluated by ELISA method. The results show that after cyclic stretch to the scleral fibroblasts of the normal sclera and the sclera after operation, the MMP-2 expression was significantly reduced and the TIMP-2 expression was significantly increased, the MMP-2 and TIMP-2 expression of the scleral fibroblasts of the fusion region after operation was no changed. It was indicated that the mechanical stimulation could regulate the MMP-2 and TIMP-2 expression of scleral fibroblasts and play an important role in the process of treating high myopia with PSR surgery.

Effect of puerarin on matrix metalloproteinase-2 in human fetal scleral fibroblasts treated with low frequency electromagnetic fields

ObjectiveTo study the effect of puerarin on matrix metalloproteinase-2 (MMP-2) gene and protein expression in human fetal scleral fibroblasts (HFSFs) exposed to extremely low frequency electromagnetic fields (ELF-EMF). MethodsCultured HFSFs were exposed to 0.2 mT ELF-EMF for 24 h. The experimental groups were divided into subgroups treated with 0, 0.1, 1 and 10 µM puerarin respectively. The expression of MMP-2 mRNA and protein were detected with real-time polymerase chain reaction and western-blot analysis respectively. ResultsMMP-2 mRNA and protein expression increased by 0.793 and 1.130 folds respectively under the exposure of ELF-EMFs at 0.2 mT flux density for 24 h. Puerarin at the concentration of 0.1 μM reversed this effect by 8.53% in mRNA and by 17.97% in protein expression (P<0.05). The effect was more prominent at higher concentrations (1 and 10 μM, P<0.01). ConclusionExposure to ELF-EMFs increased the expression of MMP-2 mRNA and protein in HFSF cells. Puerarin reversed the action to some extent in a specific concentration range. Our results implied that the puerarin might protect scleral tissue from increased expression induced by exposure to ELF-EMFs.

GABAergic Agents Modify the Response of Chick Scleral Fibroblasts to Myopic and Hyperopic Eye Cup Tissues

Purpose: GABA antagonists inhibit experimental myopia in chick and GABA receptors have been localized to chick sclera and the retinal pigment epithelium (RPE). The RPE and the choroid alter scleral DNA and glycosaminoglycan (GAG) content in vitro; opposite effects have been observed for tissues from myopic and hyperopic eyes. The aim was to determine the effect of GABAergic agents on the DNA and GAG content of chick scleral fibroblasts directly and in co-culture with ocular tissues from myopic and hyperopic chick eyes.Materials and Methods: Primary cultures of fibroblastic cells expressing vimentin and α-smooth muscle actin were established. GABAergic agents were added separately (i) to the culture medium of the scleral cells and (ii) to the culture medium of the scleral cells with the addition of posterior eye cup tissue (retina, RPE, retina + RPE, choroid + RPE) to cell culture inserts. Ocular tissues were obtained from chick eyes wearing + 15D (lens-induced hyperopia, LIH) or −15D lenses (lens-induced myopia, LIM) for three days (post-hatch day 5–8) (n = 12). GAG and DNA content of scleral fibroblasts were measured.Results: GABA agents had a small direct effect on scleral cell GAG and DNA content but a larger effect was measured when GABA agents were added to the culture medium with myopic and hyperopic RPE and choroid + RPE tissues. GABA agonists increased (p = 0.002) whereas antagonists decreased (p = 0.0004) DNA content of scleral cells; effects were opposite for scleral GAG content. GABA agents significantly altered the effect of both LIM and LIH tissues (p = 0.0005) compared to control; the effects were greater for LIM tissue versus LIH tissue co-culture (p = 0.0004).Conclusion: GABAergic agents affect the DNA and GAG content of scleral fibroblasts both directly and when co-cultured with ocular tissues. GABA antagonists that prevent myopia development in chick model could act via a scleral mechanism utilizing the RPE/choroid.

CAMP Level Modulates Scleral Collagen Remodeling, a Critical Step in the Development of Myopia

The development of myopia is associated with decreased ocular scleral collagen synthesis in humans and animal models. Collagen synthesis is, in part, under the influence of cyclic adenosine monophosphate (cAMP). We investigated the associations between cAMP, myopia development in guinea pigs, and collagen synthesis by human scleral fibroblasts (HSFs). Form-deprived myopia (FDM) was induced by unilateral masking of guinea pig eyes. Scleral cAMP levels increased selectively in the FDM eyes and returned to normal levels after unmasking and recovery. Unilateral subconjunctival treatment with the adenylyl cyclase (AC) activator forskolin resulted in a myopic shift accompanied by reduced collagen mRNA levels, but it did not affect retinal electroretinograms. The AC inhibitor SQ22536 attenuated the progression of FDM. Moreover, forskolin inhibited collagen mRNA levels and collagen secretion by HSFs. The inhibition was reversed by SQ22536. These results demonstrate a critical role of cAMP in control of myopia development. Selective regulation of cAMP to control scleral collagen synthesis may be a novel therapeutic strategy for preventing and treating myopia.

A Magnetic Bead-Based Method for Mouse Trabecular Meshwork Cell Isolation

Mice have been used widely for glaucoma research. However, due to the small size of the mouse eye, it is difficult to dissect mouse trabecular meshwork (MTM) tissues and establish MTM cell strains. To circumvent this problem, we took advantage of the phagocytic property of trabecular meshwork (TM) cells, and developed a novel magnetic bead-based method that enables us to isolate pure MTM cells.After anesthesia, up to 2 μL of fluorescent or magnetic microbeads were injected intracamerally into the mouse eyes. To study the distribution and localization of the beads, mice were sacrificed 1 to 7 days after injection, and eyes were enucleated for fluorescent or transmission electron microscopy (TEM) study, respectively. To isolate MTM cells, anterior segments injected with magnetic beads were dissected from 10 to 15 sterilized mouse eyes 7 days after injection. The tissues were digested with collagenase A and purified by using a magnetic field as well as repeated washing.TEM studies showed that the magnetic beads were located in the mouse TM, but not in corneal or scleral fibroblast cells. Cultured MTM cells were similar morphologically to human TM cells. MTM cells expressed TM markers, including collagen IV, laminin, and α-smooth muscle actin. Also, MTM cells treated with 100 nM dexamethasone showed increased formation of cross-linked actin networks and induction of myocilin expression.The magnetic bead-based method is efficient for isolating MTM cells with minimal microdissection techniques required. It will be a useful approach for isolating TM cells from small animals for glaucoma research.

Coherent Anti-Stokes Raman Scattering (CARS) Microscopy: A Novel Technique for Imaging the Retina

To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques. Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm. The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes. CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.

THE EFFECTS OF AFLIBERCEPT ON THE VIABILITY AND METABOLISM OF OCULAR CELLS IN VITRO

To investigate the effects of the vascular endothelial growth factor-neutralizing agent aflibercept on primary cultures of human trabecular meshwork cells (hTMC), human scleral fibroblasts (hFibro), and a retinal pigment epithelial cell line (ARPE-19). Various concentrations of aflibercept were incubated with confluent cell cultures for 24 hours. Ranibizumab was used as an active control for comparison. Assays of cellular metabolism (MTT assay) and cell viability (calcein dye uptake) were performed. Compared with untreated controls (100% live), a 24-hour exposure to 1 mg/mL aflibercept had no significant effect on cell viability in hTMC (100.1 ± 1.7%), hFibro (102.4 ± 2.4%), or ARPE-19 (99.3 ± 3.9%) cells. Aflibercept vehicle controls also had no detrimental effect. Aflibercept (1 mg/mL) had no statistically significant effect on metabolic activity in hTMC (84.3 ± 10.2%), hFibro (102.7 ± 4.3%), and ARPE-19 (104.6 ± 12.6%) cells. When compared side-by-side in ARPE-19 cells, aflibercept and the anti-vascular endothelial growth factor agent ranibizumab had no toxicity at the highest concentration tested (1 mg/mL). The authors' data reveal that concentrations of aflibercept in the range expected to occur in the human vitreous after intraocular injection are not harmful in an in vitro cell assay.

Exome sequencing reveals CCDC111 mutation associated with high myopia

Myopia is a refractive error of the eye that is prevalent worldwide. The most extreme form, high myopia, is usually associated with other ocular disorders such as retinal detachment, macular degeneration, cataract, and glaucoma, and is one of leading causes of blindness. The etiology is complex and has not been fully elucidated. In this study, we identified a novel missense variant of the CCDC111 gene (NM_152683.2: c.265T>G; p.Y89D) in a high myopia family by exome sequencing. The variant was identified in 4 patients from an additional 270 sporadic high myopia patients, but not found in 270 controls. The amino acid is highly conserved across species, and variants giving rise to amino acid substitutions are predicted to be functionally damaging. The CCDC111 gene was ubiquitously expressed in primary cell cultures from human eye tissue, including corneal epithelial cells, choroidal melanoma cells, scleral fibroblasts, retinal epithelial cells, retinal Müller cells, and lens capsule epithelial cells. In summary, our results suggested that the CCDC111 may be a susceptibility gene for high myopia.

Altered TGF-β2 and bFGF expression in scleral desmocytes from an experimentally-induced myopia guinea pig model

To determine changes in expression of transforming growth factor β-2 (TGF-β2) and basic fibroblast growth factor (bFGF) in scleral desmocytes from anterior and posterior portions of experimentally-induced myopic eyes of guinea pigs. Three groups (n = 10) of 2-week-old guinea pigs were used to develop concave lens-induced myopia (LIM) in one eye via the out-of-focus method for 6, 15, or 30 days respectively, while the other eye in each guinea pig served as the self-control (SC). After myopia induction, lenses were removed, and scleral fibroblasts were cultured and passaged twice. TGF-β2 and bFGF expression levels of scleral desmocytes in LIM and SC groups were compared by immunocytochemistry, quantitative real-time PCR (qRT-PCR) and Western blot analyses. The TGF-β2 expression of the anterior portion of the sclera in the LIM group was significantly higher at 15 days, and at its highest at 30 days after myopia induction compared with the SC group (P < 0.05). The TGF-β2 staining of the posterior sclera in the LIM group began to rise significantly at 6 days, peaked at 15 days and remained significantly higher than that of the anterior part, as well as the SC group, even at 30 days after myopia induction (P < 0.05). BFGF levels in scleral desmocytes from the anterior and posterior regions in the LIM group were both significantly lower than those of the SC group at all time points after myopia induction (P < 0.05). Furthermore, as the myopia progressed, bFGF expression in the anterior and posterior sclera in the LIM group gradually and statistically significantly decreased compared with the SC group (P < 0.05); however, no significant differences were observed between the anterior and posterior parts in the LIM group at any time after myopia induction (P > 0.05). All these results were consistent at the mRNA and protein levels. During myopia development in lens-induced guinea pigs, the increase in TGF-β2 activity of scleral desmocytes initiated at the posterior pole. Along with the induction time, the TGF-β2 activity in all scleral desmocytes became elevated. By contrast, the bFGF activity showed a general decline in all scleral desmocytes, rather than mainly in the posterior pole. These results imply that expression of TGF-β2 in scleral desmocytes plays a direct role, while that of bFGF exerts an indirect role in myopia development.

Zeaxanthin Induces Apoptosis in Human Uveal Melanoma Cells through Bcl-2 Family Proteins and Intrinsic Apoptosis Pathway

The cytotoxic effects of zeaxanthin on two human uveal melanoma cell lines (SP6.5 and C918) and related signaling pathways were studied and compared to effects on normal ocular cells (uveal melanocytes, retinal pigment epithelial cells, and scleral fibroblasts). MTT assay revealed that zeaxanthin reduced the cell viability of melanoma cells in a dose-dependent manner (10, 30, and 100 μM), with IC50 at 40.8 and 28.7 μM in SP6.5 and C918 cell lines, respectively. Zeaxanthin did not affect the viability of normal ocular cells even at the highest levels tested (300 μM), suggesting that zeaxanthin has a selectively cytotoxic effect on melanoma cells. Zeaxanthin induced apoptosis in melanoma cells as indicated by annexin V and ethidium III flow cytometry. Western blot analysis demonstrated that zeaxanthin decreased the expression of antiapoptotic proteins (Bcl-2 and Bcl-xL) and increased the expression of proapoptotic proteins (Bak and Bax) in zeaxanthin-treated melanoma cells. Zeaxanthin increased mitochondrial permeability as determined by JC-1 fluorescein study. Zeaxanthin also increased the level of cytosol cytochrome c and caspase-9 and -3 activities, but not caspase-8, as measured by ELISA assay or colorimetric assay. All of these findings indicate that the intrinsic (mitochondrial) pathway is involved in zeaxanthin-induced apoptosis in uveal melanoma cells.

Distribution of bone morphogenetic protein receptors in human scleral fibroblasts cultured in vitro and human sclera.

To investigate the distribution of bone morphogenetic protein receptors (BMPRs) in human scleral fibroblsasts (HSFs) and in human sclera. Primary HSFs were cultured in vitro. The mRNA levels of BMP-2 and BMPRs in HSFs were assayed by reverse transcription-polymerase chain reaction (RT-PCR). The protein distributions of BMP-2 and BMPRs in HSFs were further detected by immunocytofluorescence and western blot. Their protein expression was also detected in frozen human posterior scleral sections by immunohistofluorescence. BMP-2 and BMPRs were expressed in both HSFs and human sclera not only at mRNA level but also at protein level. The expressions of BMPRIA and BMPRII were higher than that of BMPRIB in the cytoplasm and cell membrane of HSFs in vitro. Western blot further verified the results of immunocytofluorescence. In human sclera, BMP2, BMPR IB and BMPR II were found to be expressed in the cytomatrix of HSF, and weak signal was detected about BMPRIA. BMP-2 and all three subtypes of BMPRs were found in HSFs and may play a role in scleral remodeling.

Comparative effects of posterior eye cup tissues from myopic and hyperopic chick eyes on cultured scleral fibroblasts

The role of individual ocular tissues in mediating changes to the sclera during myopia development is unclear. The aim of this study was to examine the effects of retina, RPE and choroidal tissues from myopic and hyperopic chick eyes on the DNA and glycosaminoglycan (GAG) content in cultures of chick scleral fibroblasts. Primary cultures of fibroblastic cells expressing vimentin and α-smooth muscle actin were established in serum-supplemented growth medium from 8-day-old normal chick sclera. The fibroblasts were subsequently co-cultured with posterior eye cup tissue (full thickness containing retina, RPE and choroid) obtained from untreated eyes and eyes wearing translucent diffusers (form-deprivation myopia, FDM) or −15D lenses (lens-induced myopia, LIM) for 3 days (post-hatch day 5–8) (n = 6 per treatment group). The effect of tissues (full thickness and individual retina, RPE, and choroid layers) from −15D (LIM) versus +15D (lens-induced hyperopia, LIH) treated eyes was also determined. Refraction changes in the direction predicted by the visual treatments were confirmed by retinoscopy prior to tissue collection. Glycosaminoglycan (GAG) and DNA content of the scleral fibroblast cultures were measured using GAG and PicoGreen assays. There was no significant difference in the effect of full thickness tissue from either FDM or LIM treated eyes on DNA and GAG content of scleral fibroblasts (DNA 8.9 ± 2.6 μg and 8.4 ± 1.1 μg, p = 0.12; GAG 11.2 ± 0.6 μg and 10.1 ± 1.0 μg, p = 0.34). Retina from LIM eyes did not alter fibroblast DNA or GAG content compared to retina from LIH eyes (DNA 27.2 ± 1.7 μg versus 23.2 ± 1.5 μg, p = 0.21; GAG 28.1 ± 1.7 μg versus. 28.7 ± 1.2 μg, p = 0.46). Similarly, the choroid from LIH and LIM eyes did not produce a differential effect on DNA content (DNA LIM 46.9 ± 6.4 versus LIH 51.5 ± 4.7 μg, p = 0.31). In contrast, scleral fibroblast DNA was greater in co-culture with RPE from LIM eyes than the empty basket and DNA content less for co-culture with RPE from LIH eyes (LIM: 72.4 ± 6.3 μg versus empty basket: 46.03 ± 1.0 μg; p = 0.0005 and LIH: 27.9 ± 2.3 μg versus empty basket: 46.03 ± 1.0 μg; p = 0.0004). GAG content was lower with RPE from LIM eyes (LIM: 27.7 ± 0.9 μg versus empty basket: 29.5 ± 0.8 μg, p = 0.021) and was higher with RPE from LIH eyes (LIH: 33.7 ± 1.9 μg versus empty basket: 29.5 ± 0.8 μg, p = 0.010). Choroid from LIM eyes induce a relative increase in scleral GAG content e.g. (LIM: 32.5 ± 0.7 μg versus empty basket: 29.5 ± 0.8 μg, p = 0.0004) while, choroid from LIH eyes induced a relative decrease in scleral GAG content (LIH: 18.9 ± 1.2 μg versus empty basket: 29.5 ± 0.8 μg, p = 0.0034). GAG content of cells in co-culture with choroid from LIM versus LIH treated eyes was significantly different (32.5 ± 0.7 μg versus 18.9 ± 1.2 μg respectively, p = 0.0002). In conclusion, these experiments provide an evidence for a directional growth signal that is present (and remains) in the ex-vivo RPE/choroid, but that does not remain in the ex-vivo retina. The identity of this factor(s) that can modify scleral cell DNA and GAG content requires further research.

Effect of glucagon on proliferation of scleral fibroblast cells of guinea pig in vitro and its mechanism

Background The occurrence of experimental myopia may be related to glucagon,and within the range of certain concentration,glucagon may inhibit the development of myopia,but its exact action mechanism is not completely clear.Objective Purpose of this study was to evaluate the effects of glucagon on the proliferation of guinea pig scleral fibroblast cells(GSFCs) and the possible role of glucagon in myopic scleral remodeling.Methods The scleral tissue was obtaincd from the clean blooded guinea pig aged 15 days.GSFCs were cultured and identified with vimentin antibody,cytokeratin antibody and S-100 antibody.0,5,10,50,100,200 μg/L glucagon was added into the different cultured hole for 24 hours respectively,and the growth and proliferation (A490 value) of GSFCs was detected by MTT colorimetric assay.Then the A490 value of GSFCs was assayed in 1 day,2,3,5,7 days under the 50 μg/L glucagon action.Matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2)levels(A450 value)in GSFCs were detected by enzyme-linked immuno sorbent assay (ELISA)72 hours after the cells cultured.Results Passaged GSFCs showed the dendric array at lower density and gyrate array at the higher density with the positive response for vimentin.A490 values of GSFCs were gradually increased with the rise of glucagon concentration(F=32.340,P=0.013).When the glucagon concentration was 10-200 μg/L,the A490Value of GSFCs was higher than that without glucagon group,showing signifitant differences between them(t =5.575,6.627,16.074,12.003,P＜0.05).Under the 50 μg/L glucagon action,A490 values were significantly accented with the time lapse (Ftime =10.610,P =0.024),and the A490 values also were significantly higher than the parallel control groups without glucagon(Fgroup =9.068,P=0.039).MMP-2 level was gradually declined with the enhance of glucagon within range of 5-200 μg/L(F=153.639,P=0.036),but no significant difference was found in TIMP-2 expression(F=24.770,P=3.250).Conclusions Glucagon can promote the proliferation of GSFCs in vitro,and the synthesis of MMP-2shows a concentration-and time-dependent manner. Key words: Scleral fibroblast; Glucagon; Matrix metalloproteinase-2/tissue inhibitor of metalloproteinase-2

Effects of mechanical stimulation on viscoelasticity of rabbit scleral fibroblasts after posterior scleral reinforcement

To understand the effect of mechanical stimulation on posterior scleral reinforcement (PSR), rabbit scleral fibroblasts after PSR were subjected to stretch in vitro, and the viscoelastic behavior of scleral fibroblasts was evaluated. Three-week-old rabbits were monocularly treated by eyelid suturation randomly to prepare the experimental myopia eyes. After 60 days, the experimental myopia eyes were treated by PSR. After six months, the posterior pole scleral fibroblasts (normal sclera--group A, sclera after operation--B and fusion region of sclera and reinforcing band - group C) were isolated and cultured in vitro. The cells were subjected to cyclic stretch regimens (sine wave, 3% and 6% elongation amplitude, 0.1 Hz, 48-h duration) by an FX-4000 Tension System. The micropipette aspiration technique was used to investigate the viscoelasticity of scleral fibroblasts. The cellular viscoelasticity (E(0), E(∞) and μ) of group C was significantly lower than groups A and B (P < 0.05), and there was no significant difference between groups A and B (P > 0.05). The results show that the viscoelasticity in different regions of sclera after PSR is different. Following a 48-h stretch, the cellular viscoelastic parameters were significantly decreased when compared with the respective static groups (P < 0.05) in groups A and B. For group C, the viscoelasticity of the stretch group was significantly higher than the static control group (P < 0.05). There was no difference between the 3% and 6% stretch groups in each group (P > 0.05). The changes of viscoelasticity suggest that different regions of sclera have different responses to mechanical stimulation in the process of treating high myopia by PSR and that mechanical stimulation plays an important role in the treatment of axial myopia by regulating the viscoelasticity of scleral fibroblasts.

Butein Induces Apoptosis in Human Uveal Melanoma Cells Through Mitochondrial Apoptosis Pathway

Purpose: To study the cytotoxic effects and related signaling pathways of butein on human uveal melanoma cells in vitro.Materials and Methods: Three human uveal melanoma cell lines (M17, SP6.5, and C918), retinal pigment epithelial (RPE) cells and scleral fibroblasts were treated with butein at different dosages. The effects of butein on cell viability were assessed by using the MTT assay. Cell apoptosis was determined using annexin V-FITC/ethidium homodimer III flow cytometry. Mitochondrial transmembrane potential changes were assessed by using the JC-1 fluorescent reader, cytosol cytochrome c levels, and the activities of caspase-3, -8, and -9 were measured by using an enzyme-linked immunosorbent assay or colorimetric assay.Results: Butein reduced the cell viability of cultured human uveal melanoma cells in a dose-dependent manner (10, 30, and 100 μM), with IC50 at 13.3 μM and 15.8 μM in SP6.5 and M17 cell lines, respectively. Similar effects were also found in a highly aggressive and metastatic C918 cell line (IC50 16.7 μM). Butein at lower concentrations (10–30 μM) selectively reduced the cell viability of uveal melanoma cells, without affecting cell viability of RPE cells and fibroblasts. Butein-induced apoptosis of melanoma cells, increased mitochondrial permeability and the level of cytosol cytochrome c, caspase-9 and -3 activities (but not caspase-8) in a dose-dependent manner.Conclusions: Butein has selectively potent pro-apoptotic effects on cultured human uveal melanoma cells via the intrinsic mitochondrial pathway.

Mechanical behavior of scleral fibroblasts in experimental myopia

The study aims to determine the changes in the biomechanical properties of the anterior and extreme posterior portions of experimental near-sighted eyes by examining the mechanical behavior of guinea pig scleral desmocytes, thus finding a new approach to the pathogenesis of myopia and their corresponding therapies. Guinea pigs (2weeks old) were numbered and assigned into three groups (A, B, and C) with ten guinea pigs each. Concave lens-induced myopic (LIM) animal models were prepared via the out-of-focus method. The other eye in the same guinea pig served as the self-control (SC) group. After modeling groups A, B, and C for 6, 15, and 30days respectively, the lenses from the guinea pigs in the experimental group were removed. The scleral fibroblasts in each group were cultured, and passaged twice in vitro. The micropipette aspiration technique coupled with a viscoelastic solid model was utilized to investigate the viscoelastic properties of the scleral fibroblasts in normal and myopic guinea pigs. The mechanical behavior of the scleral desmocytes of the LIM and SC groups were compared. The mechanical behavior of the scleral desmocytes was compared between the LIM and SC groups. The Young's modulus at equilibrium and the apparent cellular viscosity of the anterior portion of the sclera in the LIM group at 6days and 15days after myopic induction were not significantly different from that of the SC group (P < 0.05). However, the results for the anterior portions of the sclera in the LIM group at 30days were significantly higher than those of the LIM group at 6 and 15days, as well as those in the SC group (P < 0.05). The Young's modulus at equilibrium and the apparent cellular viscosity of the extreme posterior portions of the sclera in the LIM group at 6days after myopic induction not significantly from those of the SC group (P < 0.05). However, the results for the extreme posterior portions of the sclera in the LIM group after 15days and 30days were significantly higher than those in the LIM group at 6days and the SC group (P < 0.05). The Young's modulus at equilibrium or apparent cellular viscosity of all the anterior portions of the sclera in the LIM group were longer than those in the SC group at 30days after the induction, and the results for all the extreme posterior portions of the LIM group were larger than those of the SC group on the 15th and 30th day. Therefore, the Young's modulus and apparent viscosity of the anterior and extreme posterior portions of the sclera changed on the 15th and 30th day after induction respectively.

Ocular Lesions in Canine Mucopolysaccharidosis I and Response to Enzyme Replacement Therapy

Mucopolysaccharidosis I (MPS I) is an inherited metabolic disorder resulting from deficiency of α-L-iduronidase and lysosomal accumulation of glycosaminoglycans (GAG) in multiple tissues. Accumulation of GAG in corneal stromal cells causes corneal opacity and reduced vision. The purpose of this study was to determine the extent of ocular GAG accumulation and investigate the effectiveness of intravenous enzyme replacement therapy (ERT) on corneal GAG accumulation in dogs. Ocular tissues were obtained from 58 dogs with mucopolysaccharidosis I and four unaffected controls. Affected dogs received either low-dose ERT, high-dose ERT, or no treatment; some low-dose dogs also received intrathecal treatments. Histologic severity of corneal stromal GAG accumulation was scored. Accumulation of GAG was found in corneal stromal cells and scleral fibroblasts but not in corneal epithelium, endothelium, ciliary epithelium, choroid, retina, retinal pigment epithelium, or optic nerve. Corneal GAG accumulation increased in severity with increasing age. Although low-dose ERT did not significantly reduce corneal stromal GAG accumulation in comparison with untreated animals, high-dose ERT did result in significantly less GAG accumulation compared with the untreated dogs (adjusted P = 0.0143) or the low-dose ERT group (adjusted P = 0.0031). Intrathecal treatments did not significantly affect GAG accumulation. Dogs that began ERT shortly after birth also had significantly less (P < 0.0001) GAG accumulation in the corneal stroma than dogs with a later onset of treatment. These data suggest that high-dose, intravenous ERT is effective at preventing and/or clearing corneal stromal GAG accumulation, particularly if initiated early after birth.

Transglutaminases (TGs) in Ocular and Periocular Tissues: Effect of Muscarinic Agents on TGs in Scleral Fibroblasts

ObjectiveTo investigate the expression of transglutaminases (TGs) in the ocular surface, the eyelid margin and associated glands and to determine effect of muscarinic agents on TGs in scleral fibroblasts (SF).Materials and MethodsPrimary SFs cultured from mouse and human sclera were treated with atropine and carbachol for 5 days. Lysed cell RNA was used for real-time PCR, protein was used for Western blot analysis and TG-2 transamidase activity was measured by ELISA. Immunohistochemistry was done to determine the expression of TGases.ResultsImmunohistochemistry and western blot confirmed the expression of TGs-1, 2, 3 and 5 proteins in cultured SFs and eye tissues. Real time PCR showed TG-1, 2, 5 transcript levels to be down regulated 3 fold (p<0.05) in cultured human and mouse SFs after incubation with atropine and this was reversed by carbachol. However, TG-3 expression was increased with atropine and decreased with carbachol at all concentrations. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. TGs-1, 3, 5 were localized in the entire mouse corneal epithelium, stroma and endothelium but TG-2 was present only in the corneal subepithelium and stroma. All TGs were localized in mouse Meibomian glands however TG-2 had a weak expression.ConclusionsOur results confirm that TGs-1, 2, 3 and 5 are expressed in human SF and murine ocular tissues, eyelid and associated Meibomian glands. Real-time PCR and Western blot results showed that muscarinic antagonist down-regulates TGs-1, 2 and 5 in both cultured human and mouse SFs and upregulates TG-3. Atropine abrogated the carbachol-induced activation of SF in a dose-dependent manner. These results suggest that manipulation of TGs by way of muscarinic receptor acting drugs may be a plausible method of intervention in wound healing and scleral remodeling.

Effects of fibroblast origin and phenotype on the proliferative potential of limbal epithelial progenitor cells

The current study investigates potential differences in fibroblast phenotype across the anterior segment of the human eye with the aim to understanding factors that support the regenerative function of human limbal epithelial progenitor cells (LEPs) during wound healing. Separate cultures of fibroblasts were established from the cornea, limbus and sclera by growth in serum-supplemented medium. The resulting cultures were examined for potential differences in morphology and growth rate, as well as expression of CD34, CD45, CD90, CD141, CD271, vimentin and α-smooth muscle actin (α-sma). Finally, cultures were examined for their ability to support the growth of LEPs. While all cultures grew at a similar rate, scleral cultures often contained larger and more irregularly shaped cells which stained positive for α-sma. Western blotting confirmed a gradient of α-sma expression with lowest levels in corneal cultures. All three cultures stained positively for CD90 and vimentin, and were negative for CD34, CD45, CD141 and CD271. Only limbal or corneal irradiated fibroblasts supported the establishment of LEP cultures. While LEP colony forming efficiency and prominent expression of ABCG2, C/EBPδ and p63 was similar with either limbal or corneal fibroblasts, limbal fibroblasts supported significantly better growth. These results indicate that scleral fibroblasts have an increased capacity for myofibroblast formation which appears to negatively impact on their ability to support LEP growth. Superior growth of LEPs in the presence of limbal fibroblasts indicates a role for limbal fibroblasts in promoting the proliferation of limbal epithelium during wound healing.

Fabrication and evaluation of chitosan–gelatin based buckling implant for retinal detachment surgery

The traditional nonabsorbable silicone buckling implant (buckle) may cause some long-term complications when it is used in the retinal detachment surgery. In this study we fabricated a chitosan-gelatin based buckling implant to replace the traditional one. We evaluated its biocompatibility with human scleral fibroblasts (HSF) in vitro and its cytotoxicity with L929 cell. To evaluate elasticity and hardness of the blends buckle, the mechanical properties of the chitosan-gelatin buckle were compared with the traditional silicone buckle. The light and electron microscopy coupled with immunocytochemistry demonstrated that chitosan-gelatin blends supported the survival and growth of primarily cultured HSF without significant cytotoxic effects. MTT analysis and cell cycle analysis indicated that chitosan-gelatin blends promoted the proliferation of HSF. A preliminary in vivo implantation test indicated that chitosan-gelatin buckling implant were compatible with the surrounding tissue. The results collectively demonstrated that chitosan-gelatin blends could be a candidate biodegradable material for scleral buckling surgery.