Transglutaminases (TGs) in ocular and periocular tissues: effect of muscarinic agents on TGs in scleral fibroblasts.

OBJECTIVE: To 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 METHODS: Primary 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. RESULTS: Immunohistochemistry 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. CONCLUSIONS: Our 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.

Molecular mechanisms of muscarinic receptors in mouse scleral fibroblasts: Prior to and after induction of experimental myopia with atropine treatment.

PURPOSE: To investigate the effect of atropine on the development of spectacle lens induced myopia in the mouse and to determine if the level of mRNAs for the muscarinic receptor subtypes (M(1) - M(5)) is affected by atropine treatment. METHODS: Experimental myopia was developed in Balb/CJ (BJ) mice by placing -10 diopter spectacle lens on post-natal day 10 over the right eyes of 150 mice (n=10 in each group, 5 repetitions) for six weeks. After 2 weeks of lens wearing, the atropine group received a daily sub-conjunctival injection (10 µl) of 1% atropine sulfate and the saline group received daily 10 µl of 0.9% normal saline for 4 weeks. In addition, myopia was developed in C57BL/6 (B6) mice by placing -10 D spectacle lens on post-natal day 10 over the right eyes of 60 mice (n=10 in each group, 2 repetitions) for six weeks with and without atropine treatment. Refraction and axial length was measured at 2, 4, and 6 weeks after treatments. RT-PCR and northern blots were performed using specific primers for M(1)-M(5), and products sequenced. Real-time PCR was used to quantify message levels. RESULTS: Axial length of myopic eyes was 111% of their controls without atropine treatment and 103% of controls after atropine (p<0.01). Refraction shifted from myopic to emmetropic after atropine was administered in both pigmented and non-pigmented eyes. Corneal thickness, anterior chamber depth, corneal curvature and retinal thickness were not significantly different with and without atropine treatment (p=0.14). The lens thickness and vitreous chamber depth were significantly reduced after receiving atropine (p<0.05). Real-time PCR showed that message levels for M(1), M(3), and M(4) were upregulated in myopic sclera after atropine treatment, but M(2) and M(5) showed little change. CONCLUSIONS: The present study shows that 1% atropine reduces myopia progression in both pigmented and non-pigmented mice eyes. Axial length and vitreous chamber depth appear to be the main morphological parameters related to myopia. The results suggest that atropine may act on one or more muscarinic receptors to differentially regulate expression levels of specific receptors.

Expression of neural receptors in mouse meibomian gland.

PURPOSE: To investigate gene and protein expression profiles of neural receptors found in the mouse meibomian gland. RNA and protein levels were determined for neuropeptide Y (NPY) receptor, vasoactive intestinal peptide (VIP) receptor, substance P (SP) receptor, and muscarinic cholinergic receptor (mAChR) subtypes M1-M5 in the mouse meibomian gland. METHODS: Frozen sections of Balb/c mouse eyelids were subjected to laser capture microdissection to isolate pure samples of meibomian gland ductal and acinar cells. Real-time polymerase chain reaction, immunolabeling, and Western blot analysis for SP receptor, VIP receptor, NPY receptor, and mAChR subtypes M1-M5 were performed on meibomian gland ductal and acinar cells. RESULTS: Expression of NPY1 receptor, VIP receptor 1, SP receptor, and all 5 mAChR subtypes was found in all meibomian gland ductal and acinar cells analyzed by real-time polymerase chain reaction. Immunolabeling and Western blot analysis confirmed the presence of NPY1 receptor, VIP receptor 1, SP receptor, and all 5 mAChR subtypes in the meibomian gland. The levels were variable with the duct showing greater levels of NPY1 receptor, SP receptor, and mAChRs 1, 2, 4, and 5 than with the gland. CONCLUSIONS: VIP receptor 1, SP receptor, NPY1 receptor, and mAChR subtypes may be involved in the regulation of meibomian gland secretion. Laser capture microdissection in conjunction with gene expression analysis provides an excellent approach for studying meibomian gland cells about which relatively little is known at the molecular level.

SPARC deficiency results in improved surgical survival in a novel mouse model of glaucoma filtration surgery.

Glaucoma is a disease frequently associated with elevated intraocular pressure that can be alleviated by filtration surgery. However, the post-operative subconjunctival scarring response which blocks filtration efficiency is a major hurdle to the achievement of long-term surgical success. Current application of anti-proliferatives to modulate the scarring response is not ideal as these often give rise to sight-threatening complications. SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein involved in extracellular matrix (ECM) production and organization. In this study, we investigated post-operative surgical wound survival in an experimental glaucoma filtration model in SPARC-null mice. Loss of SPARC resulted in a marked (87.5%) surgical wound survival rate compared to 0% in wild-type (WT) counterparts. The larger SPARC-null wounds implied that aqueous filtration through the subconjunctival space was more efficient in comparison to WT wounds. The pronounced increase in both surgical survival and filtration efficiency was associated with a less collagenous ECM, smaller collagen fibril diameter, and a loosely-organized subconjunctival matrix in the SPARC-null wounds. In contrast, WT wounds exhibited a densely packed collagenous ECM with no evidence of filtration capacity. Immunolocalization assays confirmed the accumulation of ECM proteins in the WT but not in the SPARC-null wounds. The observations in vivo were corroborated by complementary data performed on WT and SPARC-null conjunctival fibroblasts in vitro. These findings indicate that depletion of SPARC bestows an inherent change in post-operative ECM remodeling to favor wound maintenance. The evidence presented in this report is strongly supportive for the targeting of SPARC to increase the success of glaucoma filtration surgery.

Expression of muscarinic receptors in human and mouse sclera and their role in the regulation of scleral fibroblasts proliferation.

PURPOSE: To determine the expression of muscarinic receptor subtypes (mAChRs) in human and mouse scleral fibroblasts (SFs), to investigate the mechanism that mediate the role mAChRs play in cell proliferation, and to explore the underlying intracellular signaling pathways involved in mouse SFs with treatment of muscarinic agents. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of mAChRs in the human and mouse sclera. Western blot analysis and immunocytochemistry were used to detect proteins of mAChRs in the cultured SFs. An immunohistochemical study was used to further detect the presence of mAChR proteins in frozen scleral sections. BrdU (5-bromo-2-deoxyuridine ) cell proliferation assay was performed to measure DNA synthesis. Enzyme linked immunosorbent assay (ELISA) was used to measure in vitro kinase activity for epidermal growth factor receptor (EGF-R), fibroblast growth factor (FGF-2), transforming growth factor (TGF)-beta1, and extracellular signal-regulated kinase (ERK)1/2. Expressions of epidermal growth factor-receptor (EGF-R); protein kinase C (PKC); Proline-rich tyrosine kinase 2 (Pyk-2), v-raf murine sarcoma viral oncogene homolog B1 (B-Raf), Rat Sarcoma (Ras), c-Jun N-terminal kinases (JNK1/2), and ERK1/2 were detected by immunoblot. RESULTS: mAChR for subtypes M(1)-M(5) were detected in both mouse and human SFs by protein, cellular, and mRNA analysis. EGF-R, PKC, Pyk-2, B-Raf, Ras, JNK1/2, and ERK1/2 were activated after treatment by agonists and antagonists, indicated by changes in phosphorylation of these proteins. Atropine abolished the carbachol-induced activation of SF cell proliferation in a concentration-dependent manner. Carbachol also activated p42/44 mitogen-activated protein kinase (MAPK) and Ras in a time-dependent manner. Muscarinic agents also modulated fibroblast growth factor expression in these cells. CONCLUSIONS: This study confirms the presence and functional role of all five mAChRs in human and mouse SFs. These results show that proliferative responses of SFs to muscarinic receptor stimulation are mediated via the activation of the classical MEK-ERK-MAPK cascade.

Effects of unilateral topical atropine on binocular pupil responses and eye growth in mice.

PURPOSE: Studies on drugs selected to target myopia development often use the vehicle-treated fellow eye as a control. However, it is not clear how much of the drug reaches the fellow eye, rendering it a potentially invalid control. Therefore, in this study, pupil responses were used to probe the effects of atropine in both eyes in mice, after unilateral topical application. In a second experiment, interocular differences in refractive development and axial eye growth were studied while atropine was applied daily to one eye. METHODS: In 20 C57BL/6 (B6) wildtype mice, a single drop of 1% atropine solution was instilled into one eye. Mice were gently restrained by holding their necks while video image processing software detected the pupil and measured its diameter at a sampling rate of 30 Hz. A bright green LED, attached to the photoretinoscope of the video camera, was flashed. Pupil responses were quantified daily over a period of 2 weeks. In another group of 24 mice, one drop of 1% atropine was applied daily for 28 days. Axial length was measured pre- and post-treatment, using low coherence interferometry (the Zeiss AC-Master). Refractive development was measured by infrared photorefraction. RESULTS: Similar to previous findings with the same device, untreated eyes displayed a pupil constriction of 24.84+/-1.73% upon stimulation with the green LED. A single drop of 1% atropine caused complete suppression with no significant recovery over the whole observation period of two weeks. The responses in the fellow eye were temporarily reduced to about 75% and then recovered towards baseline. After daily atropine application, there was significant reduction in axial length of the eyes, relative to the saline-treated fellow eyes (3.234+/-0.186 versus 3.378+/-0.176 mm, n=24, p<0.01, paired t-test) and the refractions became more hyperopic/less myopic (+13.46+/-2.15 D versus +10.06+/-2.02 D, n=24, p<0.01). CONCLUSIONS: In line with previous findings, one drop of atropine solution caused a long lasting suppression of pupil responses in the mouse eye. New data show that the transfer to the fellow eye was limited, making interocular comparisons feasible. It is also new that topical atropine reduced axial eye growth even when mice had largely normal vision.

Two models of experimental myopia in the mouse.

PURPOSE: The purpose of this study was to test the response of the mouse eye to two methods for the induction of experimental myopia. METHODS: Growth patterns of eyes were determined by axial length measurements from birth to adult in eyes of both sexes of normal mice examined on post-natal day 1 to 6 months and at 1 year. For the induction of experimental myopia, Balb/cJ mice were prepared with either unilateral lid suture or by a -10D spectacle lens placed over one eye at post-natal day 10. Other mice received a plano lens as a control for lens wear. Refraction was carried out at post-natal days of 28, 42 and 56 in lid suture and spectacle lens wear group by streak retinoscopy. Axial length was measured by a combination of video image photography, digital caliper, or Optical Low Coherence Interferometry (OLCI). Corroborative optical modeling of the mouse eye was carried out using ZEMAX ray tracing software. RESULTS: Axial length (AL) increased linearly between post-natal day 1 to day 56, plateauing at about 140 days. After 18 days of unilateral lid suture initiated 10 days after birth, the AL of experimental eyes was 3.032+/-0.003 mm, while AL in contra-lateral control eyes was 2.981+/-0.005 mm (mean+/-sem, p<0.05, n=40), after 32 days, the AL of experimental eyes was 3.290+/-0.004 mm, and the AL of control eyes was 3.104+/-0.002 mm (p<0.001, n=60). After 46 days of lid closure AL of experimental eyes was 3.592+/-0.003 mm, while AL of control eyes was 3.363+/-0.003 mm (p<0.001, n=80). Spectacle lens wear of 46 days duration increased AL in experimental eyes to 3.721+/-0.002 mm, while AL in control eyes was 3.354+/-0.003 mm (p<0.001, n=100). Refraction and ray tracing analysis substantiated the dimensional changes to be consistent with increased AL. CONCLUSIONS: Two procedures to induce experimental myopia, initiated at eye opening, produced significant myopic shifts corresponding to increases in axial lengths after 32 and 46 days of lid suture and after 46 days wearing a -10D spectacle lens.