Effects of muscarinic agents on cultured human trabecular meshwork cells

Intracellular calcium measurements were performed in cultured human trabecular meshwork cells preloaded with the cell permeant dye fura 2-AM. Fluctuations in calcium levels were then monitored with microscope-based ratio fluorometry. Carbachol increased intracellular calcium in a dose-dependent manner; as did oxotremorine-M, aceclidine, and pilocarpine. Carbachol's effect was blocked by the non-selective muscarinic antagonist atropine, as well as by muscarinic receptor subtype-selective antagonists such as pirenzepine (M1-selective), p-fHHSiD (M3-selective), and 4-DAMP (M1, M3 subtypes). Rank order of potencies for the antagonists' effects was atropine = 4-DAMP > p-fHHSiD > pirenzepine, a profile suggesting that the M3 receptor subtype is essential in the carbachol effect. Phospholipase C activity was estimated via measurement of total production of inositol phosphates in cultured human trabecular meshwork cells pre-exposed to 3H-myoinositol. In these cells, carbachol also stimulated phosphoinositide production in a dose-dependent manner, and an antagonist profile similar to that seen for calcium response was obtained when carbachol was used as the effector. The data indicate that muscarinic effects on cultured human trabecular meshwork calcium mobilization and phospholipase C activity are mediated by an M3-like receptor subtype. Therefore, the muscarinic M3 receptor may play a role in trabecular meshwork cell function(s).     

Activation of phospholipase C and guanylyl cyclase by endothelins in human trabecular meshwork cells

PURPOSE: To characterize effects of endothelins on activities of phospholipase C (PLC) and nucleotide cyclases in human trabecular meshwork (TM) cells. METHODS: Cultured simian virus 40-transformed human TM (HTM-3) or non-transformed (HTM-16) cells were used. Changes in the PLC activity were determined by assaying the production of [3H] inositol phosphates. Accumulation of cyclic GMP or cyclic AMP in cell lysate was measured by radioimmunoassay. RESULTS: Endothelin-1 (ET-1; 1 microM) stimulated PLC in HTM-16 cells, but Sarafotoxin S6c (SRTX), an ET(B) receptor subtype-selective agonist (1 microM), did not. Similar results were obtained in HTM-3 cells: ET-1, but not ET-3 or SRTX, activated PLC in a dose-dependent manner, with a calculated EC50 of 646 pM. The peptide also stimulated the accumulation of cGMP in a concentration-dependent manner with an EC50 of 37.2 pM. ET-3 or SRTX was not effective except at much higher concentrations. Both the PLC and guanylyl cyclase stimulation induced by ET-1 (10 nM) were completely inhibited by pretreating the cells with BQ-123 (<10 microM), an ET(A) receptor selective antagonist, but not by BQ-788 (10 microM), an ET(B) receptor subtype-specific antagonist. Neither ET-1 nor ET-3 stimulated adenylyl cyclase activity in HTM-3 cells at concentration as high as 1 microM. CONCLUSION: ET-1 activates PLC and guanylyl cyclase in TM cells. Potency profiles of ET receptor agonists and antagonists suggest that the ET(A) receptor subtype is involved in both actions of ET-1. The effects of the ET peptides in TM cells are interesting and could be part of the mechanism of their IOP-lowering effect.     

Lysosomal enzyme and inhibitor levels in the human trabecular meshwork

PURPOSE: To examine in the human trabecular meshwork lysosomal enzymes and one inhibitor of serine proteases that actively participate in the degradation of macromolecules into low molecular weight constituents. METHODS: Using an avidin-biotin-peroxidase technique, lysosomal proteases and alpha 1-proteinase inhibitor were examined in the trabecular meshwork of 23 human eyes with donor ages ranging from 2 to 90 years. These eyes were categorized into three age groups (< or = 20, 21 to 49, and > or = 50 years). Histochemical staining for lysosomal hydrolases was also performed on frozen sections of 20 human eyes. The staining was analyzed by an image analyzer and the levels of lysosomal proteases were further measured by biochemical assays. RESULTS: The trabecular meshwork from all the eyes stained intensely against antibodies to cathepsins B and G and alpha 1-proteinase inhibitor. The staining for elastase was weaker but evident. Image analyses revealed that the staining intensity for each protease or inhibitor was similar in all age groups. The staining in the uveal meshwork appeared to be the strongest among all the trabecular meshwork regions. Biochemical assays of tissue extracts confirmed that the enzyme and inhibitor levels were comparable among the three donor age groups. Activities of two lysosomal hydrolases, acid phosphatase and acid esterase, were also found in trabecular meshwork cells of 20 eyes. No apparent difference in enzyme activities was found with increasing age, and variation related to region was not observed. CONCLUSIONS: This study demonstrated the age-independent distribution of a variety of lysosomal enzymes and a protease inhibitor in the human trabecular meshwork. The presence of these proteins suggests a possible role in the metabolic operation of the trabecular meshwork.     

Vasoactive intestinal peptide stimulation of human trabecular meshwork cell growth

PURPOSE: To demonstrate that vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, is a growth factor of human trabecular meshwork (TM) cells in culture and in a corneoscleral explant organ culture treated with laser trabeculoplasty (LTP). METHODS: Proliferating human TM cells in cell cultures were incubated with VIP for 20 hours, followed by total cell number determination, using a Coulter counter. The percentage of proliferating TM cells was assessed, using an antibody against the proliferating cell nuclear antigen (PCNA). To test the growth effect of VIP on TM cells in situ, corneoscleral explants in organ cultures were first treated with argon LTP to initiate TM-cell proliferation and then were exposed to VIP for 48 hours. The mitotic TM cells were demonstrated immunocytochemically, using anti-PCNA in paraffin sections of the explants; and the total number of TM cells was determined after paraffin sections were counterstained by hematoxylin. RESULTS: Vasoactive intestinal peptide dose-dependently stimulated the proliferation of TM cells in cell culture. Treatment with 5 x 10(-10) M VIP resulted in a maximal increase of 40% in cell number. The effect of VIP was blocked by a VIP antagonist. The number of PCNA-stained TM cells and the total cell number in the TM in LTP-treated corneoscleral explants were increased by VIP. CONCLUSIONS: Exogenously applied VIP stimulated the proliferation of human TM cells in subconfluent cultures and in LTP-treated corneoscleral explants. In that LTP has been shown to increase the number of TM cells in situ, the growth stimulatory effect of VIP may help enhance this therapy.     

Ultrastructural changes in the trabecular meshwork of human eyes treated with corticosteroids

OBJECTIVES: To study the ultrastructure of the trabecular meshwork in human eyes with corticosteroid-induced glaucoma and to determine whether the changes noted also occur in the eyes of patients with primary open-angle glaucoma (POAG) who have been treated with corticosteroids. METHODS: The trabecular meshwork from 5 patients in whom corticosteroid-induced glaucoma was diagnosed and from 6 patients with POAG who had been treated with systemic or topical corticosteroids for months to years was investigated with light and electron microscopy. None of the eyes with POAG were considered to have corticosteroid-induced elevation of the intraocular pressure. RESULTS: Eyes with corticosteroid-induced glaucoma had the accumulation of extracellular material distinct from the sheath-derived plaques typical of POAG. A finger-printlike arranged material resembling basement membranes (FBM material), considered characteristic of corticosteroid-induced glaucoma, was found in all eyes with corticosteroid-induced glaucoma. In addition, an abnormal accumulation of densely packed, fine fibrils immediately beneath the inner wall endothelium of Schlemm's canal was present. The findings were similar among patients receiving topical or systemic treatment and among patients of different ages. In the eyes from donors with POAG who had been treated with corticosteroids, the fine fibrillar material and FBM material were present in small amounts in 3 of 6 donors and were not found in the other 3 donors. CONCLUSIONS: The extracellular material that accumulates in eyes with corticosteroid-induced glaucoma differs from that seen in eyes with POAG. Eyes with POAG exposed to long-term corticosteroid treatment did not all respond with the formation of the abnormal extracellular materials characteristic of those found in eyes with corticosteroid-induced glaucoma.     

Gene transfer to the human trabecular meshwork by anterior segment perfusion

PURPOSE: To determine whether adenovirus vectors are capable of transferring a foreign active protein to the perfused anterior segment of the human eye. METHODS: Primary cultures from the human trabecular meshwork tissue were exposed to replication-deficient adenovirus Av1LacZ4 carrying the reporter beta-galactosidase gene driven by the Rous Sarcoma Virus promoter. Anterior segments of six pairs of human eyes from normal donors were placed in organ culture and were perfused with culture medium at 2.5 microl/min constant flow. After 24 hours, one eye was injected once with 8 X 10(8) plaque-forming units (20 microl) of the viral vector, while the paired eye was injected with vehicle. Forty-eight hours (four pairs) and 7 days (two pairs) after injection, tissues were fixed, were assayed histochemically for transferred enzyme activity, and were analyzed morphologically. RESULTS: In monolayers, gene transfer occurs very efficiently in all distinct types of human outflow pathway cells. All human anterior segments injected with the adenovirus vector showed active gene transfer in cells of the outflow pathway: trabecular, juxtacanalicular, and inner wall of Schlemm's canal. Expression of the reporter enzyme was still present at 7 days after treatment. No activity was observed in any of the paired, vehicle-injected controls. Cell morphology and tissue architecture appeared normal in treated and control tissues, although some trabecular cell loss was observed in the corneoscleral and uveal regions of the perfused treated eyes. CONCLUSIONS: Adenoviral vectors were able to transfer active foreign genes into perfused, intact human trabecular meshwork.     

Expression of alternatively spliced growth factor receptor isoforms in the human trabecular meshwork

PURPOSE: Growth factors act through high-affinity cell surface receptors expressed by target cells and are critical modulators of cell function. Because aqueous humor is known to contain growth factors, these molecules may play a key role in maintaining the normal function of the human trabecular meshwork (HTM). Alternate mRNA splicing is an important mechanism used by cells to generate diverse isoforms of growth factor receptors. Although previous investigators have suggested that HTM cells may express alternative isoforms of several growth factor receptors, there have been no studies to verify these preliminary findings. The objective of this study was to determine whether cultured and ex vivo HTM cells express alternate isoforms of hepatocyte, keratinocyte, and transforming growth factor beta (TGFbeta)-II receptors and to characterize the isoform molecular sequences. METHODS: To determine whether cells within the HTM express mRNA for alternate isoforms of growth factor receptors, total RNA was isolated from several well-characterized HTM cell lines that were established from donors of various ages and from fresh ex vivo HTM tissues from healthy donors. After cDNA synthesis, polymerase chain reaction was initiated using specific primers for alternate forms of the following receptors: hepatocyte growth factor (HGFR), keratinocyte growth factor (KGFR), and transforming growth factor beta receptor II (TGFbetaR-II). Specificity and characterization of the polymerase chain reaction amplification products were determined by nucleic acid sequencing. RESULTS: Amplification products of the expected size for the growth factor isoforms were expressed in cell lines and in ex vivo tissues. Nucleic acid sequencing showed that cultured HTM cells and fresh ex vivo trabecular meshwork tissues expressed specific mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II The HGFR alternate isoform contained a 96-bp insert in the C-terminal coding region of the cytoplasmic tyrosine kinase domain. The KGFR alternate isoform is a soluble, truncated form, because it has no transmembrane or cytoplasmic domain as does the normal membrane-associated form. The TGFbetaR-II alternate isoform contained a 75-bp insert in the N-terminal coding region of the extracellular domain. CONCLUSIONS: In vitro and ex vivo HTM cells express mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II. These alternatively spliced receptor isoforms may be functional within the HTM and may play a critical role in maintaining the normal microenvironment of this important tissue.     

Functional morphology of the trabecular meshwork in primate eyes

The trabecular meshwork forms most of the resistance to aqueous humor outflow needed for maintenance of a pressure gradient between intraocular pressure of approximately 17 mmHg and venous pressure of approximately 10 mmHg. The composition of the extracellular material in the subendothelial or cribriform layer seems to be mainly responsible for outflow resistance. The aqueous humor pathways through the subendothelial layer can be influenced by ciliary muscle contraction and presumably also by contractile elements recently found both in trabecular meshwork and scleral spur. Pharmacologically induced disconnection of inner wall and cribriform cells leads to wash out of extracellular material through breaks of the endothelial lining of Schlemm's canal and to increase of outflow facility. In glaucomatous eyes the resistance to aqueous humor outflow is increased due to an increase in different forms of extracellular material deposited within the cribriform layer. The amount of this newly developed extracellular material is correlated with loss of axons in the optic nerve, indicating that a common factor is responsible for both changes. To investigate the effect of various factors on the biology of trabecular cells monolayer cultures derived from cribriform and corneoscleral trabecular meshwork have been established. The two cell lines can be differentiated because cribriform cells in vivo as in vitro stain for alphabeta-crystallin whereas the corneoscleral cells remain unstained. The effect of TGFbeta, a growth factor increased in aqueous humor of glaucomatous eyes and glycocorticoids on trabecular meshwork cells show typical changes in formation of extracellular matrix components and of stress proteins. Dexamethasone and oxidative damage also lead to increase of trabecular meshwork inducible glucocorticoid response (TIGR) protein. A mutation of the TIGR-gene family has recently been found in families with juvenile and chronic simple glaucoma. Future research has to clarify the significance of these genetic factors for the pathophysiology of glaucoma and the role of trabecular cell activity in this respect.     

Localization and possible gene expression of proteoglycan decorin in the trabecular meshwork

It is known that trabecular meshwork cells produce proteoglycans and that local production may be associated with aqueous outflow resistance. In an attempt to identify intraocular production of proteoglycan decorin in the anterior chamber angle of mammalian eyes, we conducted a Northern blot analysis and immunohistochemical studies. Northern blot analysis suggested gene expression of proteoglycan decorin in trabecular meshwork cells. Also, immunohistochemical studies using anti-decorin antibody demonstrated decorin-like immunoreactivity in the trabecular meshwork and around the Schlemm's canal. Our data demonstrate the presence of proteoglycan decorin in the outflow pathway, suggesting that decorin is a component of extracellular matrices in these regions and may be associated with outflow resistance.     

Altered beta-adrenergic regulation of Na-K-Cl cotransport in cultured smooth muscle cells from the aorta of spontaneously hypertensive rats. Role of the cytoskeleton network

To verify the hypothesis of Na-K-Cl cotransport (COTR) involvement in ion transport abnormalities as revealed in vascular smooth muscle cells (VSMC) of spontaneously hypertensive rats (SHR), we compared the rate of ouabain-insensitive, bumetanide-inhibited 86Rb influx in quiescent and growing cultures of VSMC from the aorta of SHR and normotensive (BN.lx) rats and its regulation by the cAMP signaling system. Basal COTR was not altered in quiescent cells from SHR but was decreased by 30% to 40% (P < .02) in growing SHR VSMC as compared to BN.lx rats. In quiescent BN.lx VSMC, isoproterenol inhibited COTR by 50% and induced cell shape transition of > 90% of cells, resulting in the appearance of rounded VSMC with arborized cytoplasms. In contrast, isoproterenol elicited cell shape transition in only 50% of quiescent SHR VSMC and did not modify COTR. In growing cells, it decreased COTR by 85% to 95% and altered cell morphology in > 95% of VSMC without differences between SHR and BN.lx rats. Neither inhibitors of protein kinase A (H-89 and KT-5720) nor an inhibitor of phosphoprotein phosphatase (okadaic acid) affected cell shape transition and COTR suppression in isoproterenol-treated VSMC. The COTR suppression and cytoplasm arborization was also demonstrated by the addition of cytochalasin B, a disintegrator of microfilament bundles, and staurosporine, an inhibitor of protein kinase C. The effects of these compounds on COTR and SHR and BN.lx VSMC morphology were not different. The calmodulin antagonist R24571 decreased COTR by 60% to 70% in quiescent BN.lx VSMC and did not modify this carrier in SHR VSMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of maxi-K-channels in bovine trabecular meshwork and their activation by cyclic guanosine monophosphate

PURPOSE: Electrophysiological characterization of trabecular meshwork cells and investigation of their response to elevation of cytosolic cyclic guanosine monophosphate (cGMP). METHODS: Bovine trabecular meshwork cells were cultured according to established methods and were studied, using the whole-cell and single-channel configurations of the patch-clamp technique. RESULTS: In single-channel experiments, cells expressed a channel with characteristics typical of maxi-K-channels. The channel was densely distributed in the membrane and had a high conductance of 326 +/- 4 pS (Pico Siemens) (symmetrical 150 mmol/l KCl; 37 degrees C) for potassium and negligible conductance for sodium (0.9 +/- 1 pS). The open probability could be elevated by depolarization, increasing cytosolic calcium, or adding adenosine triphosphate (1 mmol/ l). The channel could be blocked by external charybdotoxin (10(-8) mol/1), external TEA+ tetraethyl ammonium chloride (1 mmol/l) and by internal Ba2+ (10 mmol/l), whereas external Ba2+ and internal TEA+ (10 mmol/l) had no effect. In whole-cell experiments, trabecular meshwork cells displayed a strong outward conductance. Part of this conductance (35 +/- 5%) could be blocked by charybdotoxin and stimulated by ionomycin (10(-5) mol/1). Addition of 8-bromo-cGMP (10(-3) mol/1) stimulated the current to 290 +/- 57% (n = 4) of the original level, charybdotoxin led to a reduction of this current to 156 +/- 28% of the initial value. CONCLUSIONS: Trabecular meshwork cells express maxi-K-channels. These channels can be stimulated by raising internal cGMP levels and are known for their importance in smooth muscle relaxation. The results in this study supply further evidence that trabecular meshwork displays smooth muscle-like properties and contributes to the clarification of the mechanism leading to the relaxation of trabecular meshwork by nitrate and nonnitrate vasodilatators.     

Proliferation and differentiation of human trabecular osteoblastic cells on hydroxyapatite

In order to evaluate whether human osteoblastic cells differentiate normally on hydroxyapatite, we have compared the adhesion, proliferation, and differentiation of human trabecular (HT) osteoblastic cells on synthetic-dense hydroxyapatite and on standard plastic culture. We show here that initial HT cell attachment was 4-fold lower on hydroxyapatite than on plastic after 4 h of culture, and that normal cell attachment on hydroxyapatite was restored after 18 h of culture. HT cell proliferation was similar on the two substrates at 2-8 days of culture, but was lower on hydroxyapatite compared to plastic after 15 and 28 days of culture, as evaluated by DNA synthesis or cell number. HT cells cultured on both substrates produced an abundant extracellular matrix which immunostained for Type I collagen. The levels of carboxyterminal propeptide of Type I procollagen (P1CP) in the medium were lower in HT cell cultures on hydroxyapatite than on plastic. In addition, (3H)-proline incorporation into matrix proteins and the mean thickness of matrix layers were 52% and 26% lower, respectively, on hydroxyapatite compared to plastic after 4 weeks of culture, indicating that the total collagenous matrix synthesized by HT cells was lower on hydroxyapatite. However, (3H)-proline and calcium uptake expressed per cell was higher on hydroxyapatite than on plastic. The results show that human osteoblastic cells attach, proliferate, and differentiate on dense hydroxyapatite with a sequence similar to that of plastic. However, the growth of human osteoblastic cells is lower on hydroxyapatite in long-term culture, which results in a reduced amount of extracellular matrix, although matrix production per cell may be increased.     

Human and monkey trabecular meshwork accumulate alpha B-crystallin in response to heat shock and oxidative stress

PURPOSE: Oxidative stress and other forms of injury to trabecular meshwork (TM) cells may contribute to changes seen with age and primary open-angle glaucoma. This study was designed to investigate if TM expresses alpha B-crystallin, a small heat-shock protein with chaperone activity, and whether it might be overexpressed under stress conditions. METHODS: The TM from human and monkey eyes, as well as organ and primary cell cultures derived from these eyes, were investigated for alpha B-crystallin by immunohistochemistry, two-dimensional gel electrophoresis, Northern and Western blot analysis. The TM cell cultures were stressed by heat shock (44 degrees C for 15 minutes) or hydrogen peroxide (200 mumol for 1 hour). Semiquantitation of alpha B-crystallin messenger RNA (mRNA) or protein was obtained by densitometry. RESULTS: In both species, alpha B-crystallin could be detected in fresh and cultured TM by two-dimensional gel electrophoresis in conjunction with Western blot analysis. Immunohistochemistry of fresh samples showed that alpha B-crystallin was expressed predominantly in the cribriform area. Protein expression was enhanced in 4- to 7-day organ cultures. Primary cultures from human TM cells expressed two sizes (approximately 0.8 and 1.1 kb) of alpha B-crystallin mRNA in Northern blots. In monkey TM cultures, a 0.8-kb band was observed, which comigrated with lens alpha B-crystallin. In both species, heat shock caused a significant increase in alpha B-crystallin mRNA with a peak after 4 hours. An increase in alpha B-crystallin mRNA also was observed after oxidative stress; however, the onset of mRNA induction was slower. After heat shock, but not after oxidative stress, a transient change in mRNA mobility was observed. Western dot blot analysis showed a 3.4-fold increase in protein 24 hours after heat shock and a 20-fold increase after 48 hours. No constitutive mRNA expression and only a minimal increase 4 hours after heat shock could be observed in simian virus 40 transformed cell lines from human TM. CONCLUSIONS: Overexpression of alpha B-crystallin might be an important mechanism for TM to prevent cellular damage associated with various stress conditions.     

Role of protein tyrosine kinase on regulation of trabecular meshwork and ciliary muscle contractility

PURPOSE: Trabecular meshwork and ciliary muscle express properties of smooth muscle cells. The contractility of trabecular meshwork and ciliary muscle is differently modulated by various agents. To reveal contractile regulatory processes, the effects of activation and inhibition of protein tyrosine kinases (PTKs) and their interaction with other protein kinases on contractility were measured. METHODS: Measurements of isometric tension were performed on isolated bovine trabecular meshwork and ciliary muscle strips using a custom-built, electromagnetic, force-length transducer. Protein tyrosine kinase (PTK) was stimulated by epidermal growth factor (EGF) and was inhibited by genistein or tyrphostin 51. Protein kinase C (PKC) was inhibited by chelerythrine or NPC-15437 and protein kinases A and G (PKA-PKG) by H8. RESULTS: Isolated strips were precontracted by applying carbachol 10(-6) M for 30 minutes (100% carbachol maximum contraction). Inhibition of PTK evoked a maximum relaxation of 79.2+/-4.2% in trabecular meshwork and of 38.1+/-3.1% in ciliary muscle (n=8). Inhibition of PKC or PKA-PKG induced relaxations only in trabecular meshwork. When PTK and PKC or PKA-PKG were inhibited, the relaxation induced by inhibition of PTK was additive to inhibition of the other protein kinases. Stimulation of a receptor with PTK activity by EGF induced a relaxation in trabecular meshwork and a contraction in ciliary muscle precontracted by carbachol. When trabecular meshwork and ciliary muscle were activated by EGF, inhibition of PTK by genistein relaxed the cell preparations. CONCLUSIONS: Inhibition of PTK induces more prominent relaxation in trabecular meshwork than in ciliary muscle. The effects of inhibition of PTK on relaxation are independent of inhibition of PKC and PKA-PKG. The signaling cascade after activation of a tyrosine kinase receptor by EGF is differently modulated in trabecular meshwork and ciliary muscle. The effect of genistein on relaxation is probably not directly related to the EGF receptor. PTK inhibitors are possible agents for the development of novel antiglaucoma drugs.     

Experimental erbium: YAG laser photoablation of trabecular meshwork in rabbits: an in-vivo study

Photoablative laser trabecular surgery has been proposed as an outflow-enhancing treatment for open-angle glaucoma. The aim of the study was to investigate the time course of repair response following low-thermal Erbium: YAG laser trabecular ablation. In 20 anaesthetized rabbits gonioscopically controlled ab-interno photoablation of the ligamenta pectinata and underlying trabecular meshwork (TM) was performed with a single-pulsed (200 microseconds) Erbium: YAG (2.94 microns) laser. The right eye received 12-15 single laser pulses (2 mJ) delivered through an articulated zirconium fluoride fiberoptic and a 200 microns (core diameter) quartz fiber tip, the left unoperated eye served as control. At time intervals of 30 minutes, 2, 10, 30, and 60 days after laser treatment, eyes were processed for light- and scanning electron microscopy. The applied energy density of 6-4 J cm-2 resulted in visible dissection of the ligamenta pectinata and reproducible microperforations of the TM exposing scleral tissue accompanied by blood reflux from the aqueous plexus. The initial ablation zones measured 154 +/- 36 microns in depth and 45 +/- 6 microns in width. Collateral thermal damage zones were 22 +/- 8 microns. At two days post-operative, ablation craters were still blood- and fibrin-filled. The inner surface of the craters were covered with granulocytes. No cellular infiltration of the collateral thermal damage zone was observed. At 10 days post-operative, progressive fibroblastic proliferation was observed, resulting in dense scar tissue formation with anterior synechiae, proliferating capillaries and loss of intertrabecular spaces inside the range of former laser treatment at 60 days post-operative. Trabecular microperforations were closed 60 days after laser treatment in all rabbits. IOP in treated and contralateral eyes did not significantly change its level during whole period of observation. Low-thermal infrared laser energy with minimal thermal damage to collateral structures could not effectively prevent early scarring of trabecular surgery in rabbits.     

Novel trabecular meshwork inducible glucocorticoid response mutation in an eight-generation juvenile-onset primary open-angle glaucoma pedigree

OBJECTIVE: This study aimed to update a large kindred with juvenile-onset primary open-angle glaucoma (POAG) first described in 1940 and to identify the underlying genetic cause of the disease. DESIGN: Molecular genetic study of a single kindred, including clinical examination, retrospective review of clinical and family history records, linkage analysis, and mutation screening. PARTICIPANTS: The retrospective review included 957 members of a single large family. The linkage study included 40 members of 1 branch of the family in which juvenile-onset POAG is segregating in an autosomal-dominant pattern. Mutation screening included 15 at-risk family members with juvenile-onset POAG, probands of 40 families with adult-onset POAG, probands of 11 additional unrelated juvenile-onset POAG families, and 43 unrelated normal control subjects. INTERVENTION: Clinical and family history records were obtained, ophthalmologic examinations were performed, and blood samples were drawn for use in genotyping. MAIN OUTCOME MEASURES: Allele sizes of microsatellite repeat genetic markers from the vicinity of the GLC1A glaucoma gene on chromosome 1q were assigned based on size fractionation of DNA fragments generated by polymerase chain reaction (PCR). Linkage was established by the method of lod scores. Mutations were identified by determination of the DNA sequence of PCR products amplified from the trabecular meshwork inducible glucocorticoid response (TIGR) gene. Glaucoma status for purposes of linkage and mutation analysis was based on a combination of ophthalmologic examination, clinical records, family history, and previously published information. For some individuals reported in the pedigree, but not included in the genotyping studies, less information was available as presented in the text and tables. RESULTS: Autosomal-dominant POAG was confirmed or reported for 78 members of an 8-generation family. Linkage analysis showed significant evidence for linkage of juvenile-onset POAG in one branch of the family to D1S452 (maximum lod score of 6.42 at a recombination fraction of 0.00) and other markers in the vicinity of the GLC1A gene on chromosome 1q. Screening of the TIGR gene identified a mutation that results in substitution of asparagine for isoleucine at codon 477 near the carboxyterminal end of the protein. CONCLUSIONS: The authors' findings strongly suggest that the juvenile-onset POAG locus in this family is the GLC1A locus and that the underlying cause of the disease is the IIe477Asn TIGR mutation that cosegregates with juvenile-onset POAG in one branch of this large family. Lack of samples from deceased individuals prevented the authors from determining whether reported adult-onset cases in this family could also be attributed to the IIe477Asn TIGR mutation. Absence of the IIe477Asn TIGR mutation from other juvenile- and adult-onset POAG families implies that this TIGR mutation is not a common cause of glaucoma.