AP-2ß is required for formation of the murine trabecular meshwork and Schlemm's canal.

Previously, we have shown that Tfap2b, the gene encoding transcription factor AP-2ß, is needed for normal mouse eye development. Specifically, targeted loss of Tfap2b in neural crest cells (NCCs)1 and their derivatives, particularly the periocular mesenchyme (POM), resulted in anterior segment defects affecting the cornea and angle tissue. These defects were further associated with an increase in intraocular pressure (IOP). The present study investigates the underlying changes in embryonic and postnatal POM cell development and differentiation caused by loss of AP-2ß in the NCCs, particularly in the structures that control aqueous outflow, using Wnt1Cre+/-; Tfap2b-/lox; tdTomatolox/+ mice (AP-2ß neural crest cell knockout or AP-2ß NCC KO). Toluidine blue-stained sections and ultrathin sections stained with uranyl acetate and lead citrate were used to assess morphology and ultrastructure, respectively. Immunohistochemistry of KO and control eyes was performed at embryonic day (E) 15.5, E18.5, postnatal day (P) 1, P7 and P14 using phospho-histone H3 (PH3), α-smooth muscle actin (α-SMA), myocilin and endomucin antibodies, as well as a TUNEL assay. Conditional deletion of AP-2ß in the NCC-derived POM resulted in defects that appeared during both embryogenesis and postnatal stages. Fate mapping of the knockout cells in the mutants revealed that the POM migrated appropriately into the eye during embryogenesis. However, during postnatal stages a significant reduction in POM proliferation in the angle region was observed in the mutants compared to controls. This was accompanied by a lack of expression of appropriate trabecular meshwork and Schlemm's canal markers. This is the first study to show that AP-2ß is required for development and differentiation of the trabecular meshwork and Schlemm's canal. Together, these defects likely contributed to the elevated intraocular pressure (IOP) previously reported in the AP-2ß NCC KO mice.

Feasibility, compliance, and efficacy of a randomized controlled trial using vibration in pre-pubertal children.

OBJECTIVE: Interventions utilizing vibration may increase bone mass and size which may reduce forearm fractures in children. This randomized controlled pilot trial tested the feasibility, compliance and efficacy of forearm loading regimes in an after-school program in pre-pubertal children aged 6-10 years. METHODS: A 12-week randomized controlled trial incorporated high (HMMS; N=10) and low (LMMS; N=10) magnitude mechanical stimulation vibration, floor exercises (N=9), and controls (N=10). Radial bone measures by DXA and pQCT were compared at the end of intervention (12-weeks) and 4-months post-intervention (4-months post). RESULTS: Percent changes were significantly greater in floor vs. control for ultra-distal areal BMD by DXA at 12-weeks (1%[-2,5] vs.-5%[-8,-2] respectively, p=0.02) and 4-months post (5%[1,8] vs -2%[-5,2], p=0.03) and in HMMS vs. controls for trabecular vBMD by pQCT at 12-weeks (4%[0, 8], vs. -8% [-14, -2], p=0.02). Children exposed to HMMS showed positive changes in cortical BMC, area, and cortical vBMD after 12 weeks that remained 4 months post-intervention. Children exposed to floor exercise showed positive changes in cortical BMC, area, and periosteal circumference 4-months post-intervention. Controls had decreased trabecular BMD, but increased bone area and periosteal circumference. CONCLUSIONS: Exposure to floor exercise and HMMS increased trabecular aBMD and vBMD in the radius.

Cyp1b1 mediates periostin regulation of trabecular meshwork development by suppression of oxidative stress.

Mutation in CYP1B1 has been reported for patients with congenital glaucoma. However, the underlying mechanisms remain unknown. Here we show increased diurnal intraocular pressure (IOP) in Cyp1b1-deficient (Cyp1b1(-/-)) mice. Cyp1b1(-/-) mice presented ultrastructural irregular collagen distribution in their trabecular meshwork (TM) tissue along with increased oxidative stress and decreased levels of periostin (Postn). Increased levels of oxidative stress and decreased levels of Postn were also detected in human glaucomatous TM tissues. Furthermore, Postn-deficient mice exhibited TM tissue ultrastructural abnormalities similar to those of Cyp1b1(-/-) mice. Administration of the antioxidant N-acetylcysteine (NAC) restored structural abnormality of TM tissue in Cyp1b1(-/-) mice. In addition, TM cells prepared from Cyp1b1(-/-) mice exhibited increased oxidative stress, altered adhesion, and decreased levels of Postn. These aberrant cellular responses were reversed in the presence of NAC or by restoration of Cyp1b1 expression. Cyp1b1 knockdown or inhibition of CYP1B1 activity in Cyp1b1(+/+) TM cells resulted in a Cyp1b1(-/-) phenotype. Thus, metabolic activity of CYP1B1 contributes to oxidative homeostasis and ultrastructural organization and function of TM tissue through modulation of Postn expression.

Effects of vitamin K on the morphometric and material properties of bone in the tibiae of growing rats.

Suboptimal vitamin K nutriture is evident during rapid growth. We aimed to determine whether vitamin K(2) (menaquinone-4 [MK-4]) supplementation is beneficial to bone structure and intrinsic bone tissue properties in growing rats. Male Wistar rats (5 weeks old) were assigned to either a control diet (n = 8) or an MK-4-supplemented diet (22 mg d(-1) kg(-1) body weight, n = 8). After a 9-week feeding period, we determined the serum concentration ratio of undercarboxylated osteocalcin to γ-carboxylated osteocalcin and the urinary deoxypyridinoline level. All rats were then euthanized, and their tibiae were analyzed by micro-computed tomography for trabecular architecture and synchrotron radiation micro-computed tomography for cortical pore structure and mineralization. Fourier transform infrared microspectroscopy and a nanoindentation test were performed on the cortical midlayers of the anterior and posterior cortices to assess bone tissue properties. Neither body weight nor tibia length differed significantly between the 2 groups. Dietary MK-4 supplementation decreased the ratio of undercarboxylated osteocalcin to γ-carboxylated osteocalcin but did not affect deoxypyridinoline, indicating a positive effect on bone formation but not bone resorption. Trabecular volume fraction and thickness were increased by MK-4 (P < .05). Neither the cortical pore structure nor mineralization was affected by MK-4. On the other hand, MK-4 increased mineral crystallinity, collagen maturity, and hardness in both the anterior and posterior cortices (P < .05). These data indicate the potential benefit of MK-4 supplementation during growth in terms of enhancing bone quality.

Lmx1b is required for murine trabecular meshwork formation and for maintenance of corneal transparency.

Studies of Lmx1b have shown that it is required for anterior segment formation during embryonic development and that reduction of Lmx1b may contribute to elevated intraocular pressure in the adult. However, whether Lmx1b is required for formation of anterior segment tissues that are associated with regulation of intraocular pressure has not been addressed due to the perinatal lethality of Lmx1b null allele. Here we use conditional deletion strategies to circumvent perinatal lethality. Our results indicate that Lmx1b is required in neural crest-derived periocular mesenchyme for formation of anterior segment tissues, including trabecular meshwork, a critical regulator of intraocular pressure. Furthermore, we show that Lmx1b is essential to maintain proper functioning of those tissues in the adult. Taken together, our results are the first to link a specific transcription factor to trabecular meshwork formation and the first to demonstrate specific requirements for Lmx1b in maintaining the integrity of adult anterior segment.

Cultured porcine trabecular meshwork cells display altered lysosomal function when subjected to chronic oxidative stress.

PURPOSE: To investigate the effects of chronic oxidative stress on lysosomal function in trabecular meshwork (TM) cells. METHODS: Confluent cultures of porcine TM cells were grown for 2 weeks in physiological (5% O(2)) or hyperoxic conditions (40% O(2)) in the presence or absence of the protease inhibitor leupeptin (10 microM). The following parameters were quantified using the fluorogenic probes indicated within parentheses: autofluorescence, intracellular reactive oxygen species (ROS; H(2)DCFDA), mitochondrial membrane potential (JC-1), mitochondrial content (Mitotracker Red; Invitrogen-Molecular Probes, Eugene, OR), lysosomal content (acridine orange and Lysotracker Red [Invitrogen-Molecular Probes]), autophagic vacuole content (MDC), SA-beta-galactosidase (FDG), and cathepsin activities (z-FR-AMC). Cathepsin levels were quantified by qPCR and Western blot analysis. Ultrastructural analysis was performed by transmission electron microscopy. RESULTS: Prolonged exposure of porcine TM cells to a hyperoxic environment led to an increase in ROS production and oxidized material. Electron micrographs revealed the cytoplasmic accumulation of lipofuscin-loaded lysosomes. Augmented lysosomal and autophagic vacuole content was confirmed with specific fluorophores. The mRNA and protein levels of several cathepsins were upregulated with oxidative stress. This upregulated expression did not correlate with increased lysosomal activity. CONCLUSIONS: The results indicate that chronic exposure of TM cells to oxidative stress causes the accumulation of nondegradable material within the lysosomal compartment, leading to diminished lysosomal activity. Since the lysosomal system is responsible for the continuous turnover of cellular organelles, impaired lysosomal activity may lead to progressive failure of cellular TM function with age.

Enhancement of the adolescent murine musculoskeletal system using low-level mechanical vibrations.

Mechanical signals are recognized as anabolic to both bone and muscle, but the specific parameters that are critical to this stimulus remain unknown. Here we examined the potential of extremely low-magnitude, high-frequency mechanical stimuli to enhance the quality of the adolescent musculoskeletal system. Eight-week-old female BALB/cByJ mice were divided into three groups: baseline controls (BC, n = 8), age-matched controls (AC, n = 12), and whole body vibration (WBV, n = 12) at 45 Hz (0.3 g) for 15 min/day. Following 6 wk of WBV, bone mineralizing surfaces of trabeculae in the proximal metaphysis of the tibia were 75% greater (P < 0.05) than AC, while osteoclast activity was not significantly different. The tibial metaphysis of WBV mice had 14% greater trabecular bone volume (P < 0.05) than AC, while periosteal bone area, bone marrow area, cortical bone area, and the moments of inertia of this region were all significantly greater (up to 29%, P < 0.05). The soleus muscle also realized gains by WBV, with total cross-sectional area as well as type I and type II fiber area as much as 29% greater (P < 0.05) in mice that received the vibratory mechanical stimulus. The small magnitude and brief application of the noninvasive intervention emphasize that the mechanosensitive elements of the musculoskeletal system are not necessarily dependent on strenuous, long-term activity to initiate a structurally relevant response in the adolescent musculoskeletal system. If maintained into adulthood, the beneficial structural changes in trabecular bone, cortical bone, and muscle may serve to decrease the incidence of osteoporotic fractures and sarcopenia later in life.

Hormonal and biochemical determinants of trabecular microstructure at the ultradistal radius in women and men.

CONTEXT: Using high-resolution three-dimensional peripheral quantitative computed tomography (3D-pQCT) imaging, we recently described sex and age effects on bone microstructure at the ultradistal radius in men and women. Although bone volume/tissue volume decreased with age in both sexes, changes in trabecular number (TbN) and thickness (TbTh) in men were complex, with evidence for conversion of thick trabeculae into more numerous, thinner trabeculae in young men. OBJECTIVE: Our objective was to define the relationship between hormonal and bone turnover variables and trabecular microstructure at the ultradistal radius. DESIGN, SETTING, AND PARTICIPANTS: We conducted a population-based, cross-sectional study in the general community that included 205 women and 269 men, aged 21-97 yr. MAIN OUTCOME MEASURES: We measured correlation of bone volume/tissue volume, TbN, TbTh, and trabecular separation with hormonal and bone turnover variables. RESULTS: In young men (20-39 yr), TbTh and TbN were associated with serum IGF-I levels (r = 0.31, P < 0.05 and r = -0.35, P < 0.01, respectively). No associations were found between sex steroid levels (bioavailable estradiol or testosterone) or biochemical markers of bone turnover and trabecular parameters in young men or women. By contrast, in elderly men and women (>60 yr), sex steroids were the most consistently associated with trabecular microstructure, and bone turnover markers were variably associated with these parameters. CONCLUSIONS: In young men, the apparent conversion of thick trabeculae into more numerous, thinner trabeculae is most closely associated with declining IGF-I levels. By contrast, sex steroids are the major hormonal determinants of trabecular microstructure in elderly men and women.

Schlemm's canal and the collector channels at different developmental stages in the human eye.

We studied Schlemm's canal (SC) and other posttrabecular structures of aqueous humor outflow in eyes from human fetuses (at 24, 26, 33 and 36 weeks of gestation), eyes from normal children (2 months and 8 years old) and normal adult eyes, using mesoscopy/diaphanization and light microscopy. This study points to the presence of a collector canal parallel to the outer wall of SC (the outer collector). At 24 weeks of gestation, the SC was observable throughout the entire circumference (360 degrees) and the anlage of the outer collector was present. At 26 weeks of gestation, the outer collector was still growing. At 33 weeks of gestation, the outer collector was parallel to the SC and had become visible in some sectors. At 36 weeks, the SC and the outer collector were clearly defined and connected by intercanal links. The intrascleral plexus was growing. At 2 months, the deep intrascleral plexus and the episcleral plexus continued to grow, developing by sectors. At 8 years of age, the same structures corresponded to those in adults. The outer collector and the intrascleral plexuses were divided into sectors not greater than 120 degrees.

Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells.

MYOC encoding a 55kDa secretory glycoprotein named myocilin is closely linked to primary open-angle glaucoma (POAG). To understand a role played by MYOC in glaucoma, we examined the cellular fate of various mutant myocilins that were adenovirally expressed in human trabecular meshwork cells. Most myocilins with mutations such as G364V, Q368X, K423E, Y437H, and I477N were intrinsically stable, and appeared to have interactions with wild-type myocilin but not with stromelysin and thereby selectively inhibited the secretion of the former protein. The myocilins expressed were identified to be concentrated into fine punctate aggregates in endoplasmic reticulum, but never developed into the formation of aggresomes. In endoplasmic reticulum, the accumulation of the myocilins resulted in the upregulation of 78kDa glucose-regulated protein and protein disulfide isomerase. In addition, the expression of the myocilins led to deformed cellular morphology and diminished cell proliferation, an effect postulated to result in the dysfunction of trabecular cells that could be a cause of glaucoma. Therefore, our results support the statement that gain of function rather than haploinsufficiency is a critical mechanism for POAG in individuals with mutations on MYOC.

The mouse anterior chamber angle and trabecular meshwork develop without cell death.

BACKGROUND: The iridocorneal angle forms in the mammalian eye from undifferentiated mesenchyme between the root of the iris and cornea. A major component is the trabecular meshwork, consisting of extracellular matrix organized into a network of beams, covered in trabecular endothelial cells. Between the beams, channels lead to Schlemm's canal for the drainage of aqueous humor from the eye into the blood stream. Abnormal development of the iridocorneal angle that interferes with ocular fluid drainage can lead to glaucoma in humans. Little is known about the precise mechanisms underlying angle development. There are two main hypotheses. The first proposes that morphogenesis involves mainly cell differentiation, matrix deposition and assembly of the originally continuous mesenchymal mass into beams, channels and Schlemm's canal. The second, based primarily on rat studies, proposes that cell death and macrophages play an important role in forming channels and beams. Mice provide a potentially useful model to understand the origin and development of angle structures and how defective development leads to glaucoma. Few studies have assessed the normal structure and development of the mouse angle. We used light and electron microscopy and a cell death assay to define the sequence of events underlying formation of the angle structures in mice. RESULTS: The mouse angle structures and developmental sequence are similar to those in humans. Cell death was not detectable during the period of trabecular channel and beam formation. CONCLUSIONS: These results support morphogenic mechanisms involving organization of cellular and extracellular matrix components without cell death or atrophy.

Ultrastructural studies of primary congenital glaucoma in rabbits.

BACKGROUND: The cause of congenital glaucoma is unknown. METHODS: To determine whether the site of impaired aqueous outflow is the entrance to the trabecular meshwork (TM), within the TM, the aqueous drainage plexus, or a combination thereof, the process of TM development was examined by scanning and transmission electron microscopy on postnatal day 3 and weeks 1, 2, 3, 4, and 6 in New Zealand rabbits homozygous for the buphthalmic (bu/bu) gene compared with age-matched controls. RESULTS: Openings to the entrance of the TM in congenital glaucoma were observed, and there was no evidence of an endothelial membrane occluding aqueous flow to the TM. The morphology of the congenital glaucoma TM was abnormal in all bu/bu rabbits by 2 weeks and was characterized by a smaller entrance to the TM at the iris base, smaller intertrabecular openings within and between the trabecular lamellae, and at 6 weeks, iris pillars with extensive lateral extensions in the angle recess. Most intertrabecular spaces were open, however, the inner intertrabecular spaces adjacent to the aqueous plexus were compressed. CONCLUSION: These results suggest the development of congenital glaucoma, which involves a mutation in an autosomal recessive gene and leads to loss of function of a gene(s) required for the differentiation of the TM.

Effect of age on superoxide dismutase activity of human trabecular meshwork.

PURPOSE: It has been hypothesized that accelerated aging of the trabecular meshwork, perhaps because of oxidative damage, is involved in the pathogenesis of primary open angle glaucoma. The authors sought to evaluate the effect of donor age on the specific activity of superoxide dismutase and catalase in normal fresh human cadaver trabecular meshwork. METHODS: Total superoxide dismutase and catalase were assayed in tissue extracts generated from fresh human trabecular meshwork. Cadaver tissue was obtained from 19 donors (18 paired) of a wide age range (30 to 91 years). The assays were performed within 6 hours of enucleation and within 36 hours of donor death. Enzyme-specific activities were calculated using protein concentration of the extract as the denominator. RESULTS: Multiple regression analysis modeled with age and time from death until the beginning of the experiment was performed. The specific activity of superoxide dismutase declined with age (P = 0.00022; r2 = 0.67). There was no effect of age on catalase specific activity (P = 0.24; r2 = 0.16). The time from donor death until the beginning of the experiment was not a significant factor (P > 0.28). CONCLUSIONS: The specific activity of superoxide dismutase, but not catalase, demonstrates an age-dependent decline in normal cadaver human trabecular meshwork. The potential role of superoxide dismutase in primary open angle glaucoma, a disorder of the aging trabecular meshwork, warrants further investigation.

Fluorescence-labeled lectins, glycoconjugates, and the development of the mouse AOP.

The development of the aqueous outflow pathway (AOP) in early postnatal mouse eyes was examined for the presence of a variety of lectin receptors using fluorescein isothiocyanate (FITC) conjugated lectins, 1 micron araldite plastic sections, and computer-aided fluorescence photography. The trabecular meshwork anlage (days 1-4) was characterized by the presence of loosely arranged cells and an extracellular matrix that exhibited intense areas of Con A- and RCA-lectin staining, and absence of WGA- and LPA-lectin staining. By day 6, trabecular meshwork LPA- and WGA-positive materials were observed as focal areas of staining. By day 10, LPA- and WGA-positive materials were present as diffuse areas of staining, as the AOP differentiated into an organized and functional biological filter. The age-dependent pattern of LPA- and WGA-positive materials indicated that there were time-dependent points in the synthesis of glycoconjugates in the developing AOP. The results suggest: The composition and/or conformation of the glycoconjugates on cells and extracellular matrix changed as the AOP differentiated into a functional tissue. The use of FITC lectins as biological markers for studies of the AOP provided information on the potential role of glycoconjugates in the development of the normal AOP. Modification in the type, amount, and distribution of glycoconjugates may provide a basis for understanding the cellular mechanisms of abnormal development of the AOP, eg, congenital glaucoma.

Age-related changes in the trabecular meshwork of the normal human eye.

Specimens from 17 human eyes, ranging in age from 3 to 80 years, were subjected to morphometric studies using light microscope, transmission electron microscope and scanning electron microscope, in order to clarify the age-related changes in the normal trabecular tissue. Statistical analyses showed that the cellularity in the various regions of the trabecular meshwork significantly declined with age. The spaces corresponding to the aqueous outflow pathway in each region of the meshwork also significantly decreased with age. On the other hand, extracellular materials significantly increased in amount with age in all regions of the trabecular meshwork. However, the decrease in the cellularity and the outflow pathway spaces did not show any statistically significant regional difference. These results suggest that general narrowing of the outflow pathway spaces due to the accumulation of extracellular materials with age is the cause of the increase in aqueous outflow resistance with age, and that each region of the trabecular meshwork is equally responsible for the increased resistance.

Developmental immaturity of the trabecular meshwork in juvenile glaucoma.

We used light and electron microscopy to compare ten specimens of anterior chamber angle tissue obtained by trabeculectomy from seven patients with juvenile glaucoma (primary glaucoma with open iridocorneal angle in young humans with specimens of normal trabecular meshwork from humans of the same ages. In all cases of juvenile glaucoma, we observed a thick compact tissue consisting of cells with fine processes and extracellular substances at the anterior chamber side of Schlemm's canal and abnormal deposits of ground substances in the thick tissue. These observations strongly suggest that the thick compact tissue represents an immature development of the trabecular meshwork and may be one of the primary causes of increased intraocular pressure in juvenile glaucoma. Our studies indicate that both congenital and juvenile glaucoma occur because of developmental immaturity of the trabecular meshwork in anterior chamber angle tissue and that the more extensive the immaturity, the earlier the glaucoma will become manifest.

The development of the chamber angle in the rat eye. Morphological characteristics of developmental stages.

The pre- and postnatal development of the chamber angle region was studied in the rat. The eyes of five weight-matched littermates were investigated by light- and electron microscopy at each of the following times: prenatal days 14, 17, 18, 21; day of birth; postnatal days 5, 10, 15, 20, 40, 60, 100, 200. Major developmental stages are demonstrated and the morphological characteristics of each stage shown. Developmental periods in the rat are correlated with periods that have previously been outlined in the human (Remé and Lalive 1981). Major periods comprise the following events: I. anlage formation (day 17 prenatal - day postnatal); II. differentiation into definitive structures (days 5-10); III. specialization of definitive structures (days 10-20); IV. final components (days 20-60); V. final molding and rarefaction (days 60-100). Our data indicate that the developing rat eye is a suitable experimental model for studies on the development of the chamber angle. Moreover, this study provides baseline data for a separate investigation on morphogenetic events contributing to the remodeling processes occurring within the vertebrate chamber angle.

The occurrence of cell death during the remodelling of the chamber angle recess in the developing rat eye.

Prenatal and postnatal stages of the developing chamber angle in the rat eye were analysed qualitatively by light and electron microscopy. One eye of each of five weight-matched littermates was investigated at the following time points: prenatal days 14, 17, 18 and 21; day 0 = day of birth; postnatal days 5, 10, 15, 20, 40, 60, 100 and 200. From postnatal days 5 to 60, conspicuous amounts of necrotic cells were observed within the chamber angle area. At birth and at postnatal day 200, only a few scattered necroses were observed. Necrotic cells were found within the trabecular area, the region of Fontana's spaces and beams and the peripheral iris, the iris root, the ciliary body and the adjoining choroid. Large macrophages containing lysosomal dense bodies were found within the previously described areas and were frequently in close contact with necrotic cells. It is concluded that cell death within the developing chamber angle of the rat may represent an important event that may contribute to two morphogenetic effects: (1) the opening of large spaces of Fontana and (2) space formation within the trabecular area and uveoscleral outflow routes.