Accommodative movements of the choroid in the optic nerve head region of human eyes, and their relationship to the lens.

The ciliary muscle (CM) powers the accommodative response, and during accommodation the CM pulls the choroid forward in the region of the ora serrata. Our goal was to elucidate the accommodative movements of the choroid in the optic nerve region in humans and to determine whether these movements are related to changes in the lens dimensions that occur with aging, in the unaccommodated and accommodated state. Both eyes of 12 human subjects (aged 18-51 yrs) were studied. Homatropine (1 drop/5%) was used to relax the ciliary muscle (unaccommodated or "resting" eye) and pilocarpine was used to induce the maximum accommodative response (2 drops/4%) (accommodated eye). Images of the fundus and choroid were collected in the region of the optic nerve (ON) via Spectralis OCT (infrared and EDI mode), and choroidal thickness was determined. Ultrasound biomicroscopy (UBM; 50 MHz, 35 MHz) images were collected in the region of the lens/capsule and ciliary body. OCT and UBM images were collected in the resting and accommodated state. The unaccommodated choroidal thickness declined significantly with age (p = 0.0073, r = 0.73) over the entire age range of the subjects studied (18-51 years old). The choroidal thickness was significantly negatively correlated with lens thickness in the accommodated (p = 0.01) and the unaccommodated states (p = 0.005); the thicker the lens the thinner the choroid. Choroid movements around the optic nerve during accommodation were statistically significant; during accommodation the choroid both thinned and moved centrifugally (outward/away from the optic nerve head). The accommodative choroid movements did not decline significantly with age and were not correlated with accommodative amplitude. Measurement of the choroidal thickness is possible with the Spectralis OCT instrument using EDI mode and can be used to determine the accommodative changes in choroidal thickness. The choroidal thickness decreased with age and during accommodation. It may be that age-related choroidal thinning is due to changes in the geometry of the accommodative apparatus to which it is attached (i.e., ciliary muscle/lens complex) such that when the lens is thicker, the choroid is thinner. Accommodative decrease in choroidal thickness and stretch of the retina/choroid may indicate stress/strain forces in the region of the optic nerve during accommodation and may have implications for glaucoma.

Therapeutic potential of AAV-mediated MMP-3 secretion from corneal endothelium in treating glaucoma.

Intraocular pressure (IOP) is maintained as a result of the balance between production of aqueous humour (AH) by the ciliary processes and hydrodynamic resistance to its outflow through the conventional outflow pathway comprising the trabecular meshwork (TM) and Schlemm's canal (SC). Elevated IOP, which can be caused by increased resistance to AH outflow, is a major risk factor for open-angle glaucoma. Matrix metalloproteinases (MMPs) contribute to conventional aqueous outflow homeostasis in their capacity to remodel extracellular matrices, which has a direct impact on aqueous outflow resistance and IOP. We observed decreased MMP-3 activity in human glaucomatous AH compared to age-matched normotensive control AH. Treatment with glaucomatous AH resulted in significantly increased transendothelial resistance of SC endothelial and TM cell monolayers and reduced monolayer permeability when compared to control AH, or supplemented treatment with exogenous MMP-3.Intracameral inoculation of AAV-2/9 containing a CMV-driven MMP-3 gene (AAV-MMP-3) into wild type mice resulted in efficient transduction of corneal endothelium and an increase in aqueous concentration and activity of MMP-3. Most importantly, AAV-mediated expression of MMP-3 increased outflow facility and decreased IOP, and controlled expression using an inducible promoter activated by topical administration of doxycycline achieved the same effect. Ultrastructural analysis of MMP-3 treated matrices by transmission electron microscopy revealed remodelling and degradation of core extracellular matrix components. These results indicate that periodic induction, via use of an eye drop, of AAV-mediated secretion of MMP-3 into AH could have therapeutic potential for those cases of glaucoma that are sub-optimally responsive to conventional pressure-reducing medications.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Age-related posterior ciliary muscle restriction - A link between trabecular meshwork and optic nerve head pathophysiology.

The ciliary muscle plays a major role in controlling both accommodation and outflow facility in primates. The ciliary muscle and the choroid functionally form an elastic network that extends from the trabecular meshwork all the way to the back of the eye and ultimately attaches to the elastic fiber ring that surrounds the optic nerve and to the lamina cribrosa through which the nerve passes. The ciliary muscle governs the accommodative movement of the elastic network. With age ciliary muscle mobility is restricted by progressively inelastic posterior attachments and the posterior restriction makes the contraction progressively isometric; placing increased tension on the optic nerve region. In addition, outflow facility also declines with age and limbal corneoscleral contour bows inward. Age-related loss in muscle movement and altered limbal corneoscleral contour could both compromise the basal function of the trabecular meshwork. Further, recent studies in non-human primates show that the central vitreous moves posteriorly all the way back to the optic nerve region, suggesting a fluid current and a pressure gradient toward the optic nerve. Thus, there may be pressure and tension spikes on the optic nerve region during accommodation and these pressure and tension spikes may increase with age. This constellation of events could be relevant to glaucomatous optic neuropathy. In summary, our hypothesis is that glaucoma and presbyopia may be literally linked to each other, via the choroid, and that damage to the optic nerve may be inflicted by accommodative intraocular pressure and choroidal tension "spikes", which may increase with age.

Anteriorly located zonular fibres as a tool for fine regulation in accommodation.

PURPOSE: To describe an anteriorly located system of zonular fibres that could be involved in fine-tuning of accommodation. METHODS: Forty-six human and 28 rhesus monkey eyes were dissected and special preparations were processed for scanning electron microscopy and reflected-light microscopy. Additional series of frontal and sagittal histological and ultrathin sections were analysed in respect to the origin and insertion of anteriorly located zonules. The presence of sensory terminals at the site of the originating zonules within the connective tissue of the ciliary body was studied by immunohistochemistry. For in-vivo visualization ultrasound biomicroscopy (UBM) was performed on 12 human subjects. RESULTS: Fine zonular fibres originated from the valleys and lateral walls of the most anterior pars plicata that covers the anterior and inner circular ciliary muscle portion. These most anterior zonules (MAZ) showed attachments either to the anterior or posterior tines or they inserted directly onto the surface of the lens. At the site of origin, the course of the MAZ merged into the connective tissue fibres connecting the adjacent pigmented epithelium to the ciliary muscle. Numerous afferent terminals directly at the site of this MAZ-origin were connected to the intrinsic nervous network of the ciliary muscle. CONCLUSIONS: A newly described set of zonular fibres features the capabilities to register the tensions of the zonular fork and lens capsule. The close location and neural connection towards the circular ciliary muscle portion could provide the basis for stabilization and readjustment of focusing that serves fast and fine-tuned accommodation and disaccommodation.

Ocular surface changes in mice with streptozotocin-induced diabetes and diabetic polyneuropathy.

PURPOSE: Diabetes mellitus (DM) is a leading risk factor for corneal neuropathy and dry eye disease (DED). Another common consequence of DM is diabetic peripheral polyneuropathy (DPN). Both complications affect around 50 % of the DM patients but the relationship between DM, DED and DPN remains unclear. METHODS: In this study, we examined mice with early onset of DM and PN after streptozotocin (STZ)-induced diabetes (DPN). We compared the early morphological changes of the sciatic nerve, dorsal root and trigeminal ganglia with the changes in the ocular surface, including tear proteomic and we also investigated respective changes in the gene expressions and morphological alterations in the eye tissues involved in tear production. RESULTS: The lacrimal gland, conjunctival goblet cells and cornea showed morphological changes along with alterations in tear proteins without any obvious signs of ocular surface inflammation. The gene expression for respectively altered tear proteins i.e., of Clusterin in cornea, Car6, Adh3a1, and Eef1a1 in eyelids, and Pigr in the lacrimal gland also showed significant changes compared to control mice. In the trigeminal ganglia like in the dorsal root ganglia neuronal cells showed swollen mitochondria and, in the latter, there was a significant increase of NADPH oxidases and MMP9 suggestive of oxidative and neuronal stress. In the dorsal root ganglia and the sciatic nerve, there was an upregulation of a number of pro-inflammatory cytokines and pain-mediating chemokines. CONCLUSION: The early ocular changes in DM Mice only affect the lacrimal gland. Which, is reflected in the tear film composition of DPN mice. Due to the high protein concentration in tear fluid in humans, proteomic analysis in addition to noninvasive investigation of goblet cells and cornea can serve as a tools for the early diagnosis of DPN, DED in clinical practice. Early treatment could delay or even prevent the ocular complications of DM such as DED and PN.

Matrix metalloproteinase-3 (MMP-3)-mediated gene therapy for glaucoma.

Approximately 80 million people globally are affected by glaucoma, with a projected increase to over 110 million by 2040. Substantial issues surrounding patient compliance remain with topical eye drops, and up to 10% of patients become treatment resistant, putting them at risk of permanent vision loss. The major risk factor for glaucoma is elevated intraocular pressure, which is regulated by the balance between the secretion of aqueous humor and the resistance to its flow across the conventional outflow pathway. Here, we show that adeno-associated virus 9 (AAV9)-mediated expression of matrix metalloproteinase-3 (MMP-3) can increase outflow in two murine models of glaucoma and in nonhuman primates. We show that long-term AAV9 transduction of the corneal endothelium in the nonhuman primate is safe and well tolerated. Last, MMP-3 increases outflow in donor human eyes. Collectively, our data suggest that glaucoma can be readily treated with gene therapy-based methods, paving the way for deployment in clinical trials.

GPR40 Activation Abolishes Diabetes-Induced Painful Neuropathy by Suppressing VEGF-A Expression.

G-protein-coupled receptor 40 (GPR40) is a promising target to support glucose-induced insulin release in patients with type 2 diabetes. We studied the role of GPR40 in the regulation of blood-nerve barrier integrity and its involvement in diabetes-induced neuropathies. Because GPR40 modulates insulin release, we used the streptozotocin model for type 1 diabetes, in which GPR40 functions can be investigated independently of its effects on insulin release. Diabetic wild-type mice exhibited increased vascular endothelial permeability and showed epineural microlesions in sciatic nerves, which were also observed in naïve GPR40-/- mice. Fittingly, expression of vascular endothelial growth factor-A (VEGF-A), an inducer of vascular permeability, was increased in diabetic wild-type and naïve GPR40-/- mice. GPR40 antagonists increased VEGF-A expression in murine and human endothelial cells as well as permeability of transendothelial barriers. In contrast, GPR40 agonists suppressed VEGF-A release and mRNA expression. The VEGF receptor inhibitor axitinib prevented diabetes-induced hypersensitivities and reduced endothelial and epineural permeability. Importantly, the GPR40 agonist GW9508 reverted established diabetes-induced hypersensitivity, an effect that was blocked by VEGF-A administration. Thus, GPR40 activation suppresses VEGF-A expression, thereby reducing diabetes-induced blood-nerve barrier permeability and reverting diabetes-induced hypersensitivities.

Prolonged Sox4 expression in oligodendrocytes interferes with normal myelination in the central nervous system.

The highly related transcription factors Sox4 and Sox11 are both expressed in oligodendrocyte precursors. Yet whether they have a function in oligodendrocyte development is unknown. By overexpressing Sox4 under the control of 3.1 kb of 5' flanking sequences of the myelin basic protein gene in transgenic mice, we extended Sox4 expression in the oligodendrocyte lineage from oligodendrocyte precursors to cells undergoing terminal differentiation. As a consequence of transgene expression, mice develop the full spectrum of phenotypic traits associated with a severe hypomyelination during the first postnatal weeks. Myelin gene expression was severely reduced, and myelin dramatically thinned in several central nervous system (CNS) regions. Despite these disturbances in CNS myelination, the number of oligodendrocytic cells remained unaltered. Considering that apoptosis rates were normal and proliferation only slightly increased, oligodendrocytes likely persist in a premyelinating to early myelinating state. This shows that prolonged Sox4 expression in cells of the oligodendrocyte lineage is incompatible with the acquisition of a fully mature phenotype and argues that the presence of Sox4, and possibly Sox11, in oligodendrocyte precursors may normally prevent premature differentiation.

The ß4-Subunit of the Large-Conductance Potassium Ion Channel KCa1.1 Regulates Outflow Facility in Mice.

Purpose: The large-conductance calcium-activated potassium channel KCa1.1 (BKCa, maxi-K) influences aqueous humor outflow facility, but the contribution of auxiliary ß-subunits to KCa1.1 activity in the outflow pathway is unknown. Methods: Using quantitative polymerase chain reaction, we measured expression of ß-subunit genes in anterior segments of C57BL/6J mice (Kcnmb1-4) and in cultured human trabecular meshwork (TM) and Schlemm's canal (SC) cells (KCNMB1-4). We also measured expression of Kcnma1/KCNMA1 that encodes the pore-forming α-subunit. Using confocal immunofluorescence, we visualized the distribution of ß4 in the conventional outflow pathway of mice. Using iPerfusion, we measured outflow facility in enucleated mouse eyes in response to 100 or 500 nM iberiotoxin (IbTX; N = 9) or 100 nM martentoxin (MarTX; N = 12). MarTX selectively blocks ß4-containing KCa1.1 channels, whereas IbTX blocks KCa1.1 channels that lack ß4. Results: Kcnmb4 was the most highly expressed ß-subunit in mouse conventional outflow tissues, expressed at a level comparable to Kcnma1. ß4 was present within the juxtacanalicular TM, appearing to label cellular processes connecting to SC cells. Accordingly, KCNMB4 was the most highly expressed ß-subunit in human TM cells, and the sole ß-subunit in human SC cells. To dissect functional contribution, MarTX decreased outflow facility by 35% (27%, 42%; mean, 95% confidence interval) relative to vehicle-treated contralateral eyes, whereas IbTX reduced outflow facility by 16% (6%, 25%). Conclusions: The ß4-subunit regulates KCa1.1 activity in the conventional outflow pathway, significantly influencing outflow function. Targeting ß4-containing KCa1.1 channels may be a promising approach to lower intraocular pressure to treat glaucoma.

Structural changes of the trabecular meshwork in different kinds of glaucoma.

The morphology of the trabecular meshwork in three types of open angle glaucoma: primary open angle glaucoma (POAG), corticosteroid-induced glaucoma and pigmentary glaucoma (PG) are described. Ageing is one major risk factor for development of POAG. It is assumed that preexisting age-related changes of the trabecular meshwork (TM) play a role for the development of increased outflow resistance and intraocular pressure (IOP) in various types of glaucoma. These age-related changes in the TM develop concomitant with that of presbyopia. Therefore the functional relationship between ciliary muscle (CM) and TM and the age-related changes in morphology of the outflow system are described first. One main finding in the ageing TM concerns changes of the elastic fiber network and the anterior elastic tendons of the CM. There is an increase in thickness of the sheath of the elastic fibers. Cross-sections through these fibers with their sheath appear as extracellular plaques and were therefore termed "sheath derived plaques" (SD-plaques). Morphologically, the TM changes in POAG resemble that of the ageing TM, but in POAG there is a significant increase in SD-plaques compared to age-matched controls. This increase is due to fine fibrils and other components of the extracellular matrix (ECM) that adhere to the sheaths of the elastic fibers and their connections to the inner wall endothelium. In POAG eyes there is also a marked loss of TM cells, at places leading to fusion and thickening of trabecular lamellae. In steroid-induced glaucoma there is also an increase in fine fibrillar material in the subendothelial region of SC. In contrast to POAG eyes these fibrils do not adhere to the sheath of the elastic fibers but are deposited underneath the inner wall endothelium. The main finding in steroid-induced glaucoma is an accumulation of basement membrane-like material staining for type IV collagen. These accumulations are found throughout all layers of the TM. In pigmentary glaucoma loss of cells was more prominent than in POAG eyes. Presumably, this cell loss occurs after overload of TM cells with pigment granules. Denuded TM lamellae fuse and the TM collapses. In the subendothelial region of these collapsed TM areas an increase in ECM presumably due to underperfusion was observed. At other places SC was occluded and the cribriform region appeared disorganized. In most parts of the circumference of the eye, the TM cells contained pigment granules. Occlusion of TM spaces by pigment granules or cells loaden with pigment was not seen in eyes with PG.

The effect of TGF-beta2 on elastin, type VI collagen, and components of the proteolytic degradation system in human optic nerve astrocytes.

PURPOSE: To investigate the effect of transforming growth factor (TGF)-beta2 on the expression of (1) elastin and type VI collagen (ColVI), (2) extracellular matrix (ECM)-degrading matrix-metalloproteinases (MMPs) and regulators of their activity/activation, and (3) the involvement of connective tissue growth factor (CTGF) in the TGF-beta2-mediated regulations in cultured human type-1A and -1B optic nerve astrocytes. METHODS: Astrocytes were isolated from the optic nerves of 11 donors aged 19 to 62 years without a history of eye disease from the prelaminar (type 1B, five explants) or postlaminar (type 1A, six explants) region. Cultures of passages 3 to 5 were treated with 1 ng/mL recombinant human TGF-beta2 for 72 hours, and regulatory effects on the expression of elastin and the ColVI chains alpha1, alpha2, and alpha3; MMP-1, -2, -3, -7, -9, -12, and -13; tissue inhibitors of MMPs (TIMPs) -1, -2, and -3; plasminogen activator inhibitor 1 (PAI-1); and urokinase and tissue plasminogen activators (uPA, tPA) were initially analyzed by RT-PCR and confirmed and quantified by real-time PCR (rtPCR). The regulation of proteins was studied by Western blot analysis, and MMP-2 activity was assessed by gelatin zymography. The involvement of CTGF was tested by knockdown experiments with CTGF-small interfering (si)RNA. RESULTS: TGF-beta2 increased the expression of elastin (5X[rtPCR]/6X[WB]), ColVIalpha2 (3X/5X), ColVIalpha3 (7X/9X), MMP-2 (2X/2X), TIMP-1/-3 (1.5X/2X), and PAI-1 (8X/4X) compared to untreated controls. tPA was reduced to 0.5X. MMP-1, -3, -7, and -12 and TIMP-2 were expressed but were not responsive to TGF-beta2. MMP-9 and -13 and uPA were marginally expressed and close to the detection threshold. MMP-2 activity was significantly reduced in gelatin zymography. Transfection of CTGF-siRNA blocked TGF-beta2-mediated activation of elastin and ColVI but had no effect on MMP-2 and PAI-1 induction. Type 1A and 1B astrocytes reacted identically. CONCLUSIONS: TGF-beta2 induces expression of elastin and ColVI and thereby could contribute to the increase of type VI collagen fibers in the tissue septae and the elastotic changes typically observed in POAG. With the concurrent activation of TIMP-1 and -3 and PAI-1 and the repression of tPA, TGF-beta2 could negatively regulate the activity and activation of MMPs. This effect could further amplify ECM accumulation and elastosis.

Dependency of intraocular pressure elevation and glaucomatous changes in DBA/2J and DBA/2J-Rj mice.

PURPOSE: In this study parameters relevant for glaucoma in DBA/2J (D2J) mice were compared with those in age-matched DBA/2J-Rj (D2Rj) mice, to challenge the postulated role of D2J mice as a model for secondary high-tension glaucoma. METHODS: Genotyping for three known short nucleotide polymorphisms (SNPs) in the Tyrp1 gene and the Gpnmb gene by MALDI-TOF-MS and immunohistochemical staining for Gpnmb was performed in D2J and D2Rj mice. Twelve C57Bl/6 (B6), 8 D2Rj, and 11 D2J mice between 1 and 4 months of age were screened qualitatively and quantitatively for morphologic differences within the anterior eye segment. The IOP progression of 25 D2Rj and 18 D2J mice were investigated between 4 to 10.5 months after birth. At the end of this study, in 10 randomly selected individuals of each D2J and D2Rj cohort, correlation of IOP progression and optic nerve damage were determined in each eye. RESULTS: D2J and D2Rj strains were homozygous for both Tyrp 1 amino acid substitutions, so far only described in D2J mice. The Gpnmb(R150X) point mutation present in D2J mice was not detected in D2Rj. Accordingly, immunoreactivity (IR) for Gpnmb was present only in D2Rj and B6 eyes, but not in D2J. Compared with B6, both DBA/2 mice (D2) showed a significantly narrowed chamber angle caused by an anteriorly displaced ciliary body. IOP measurements showed an average IOP of approximately 14 mm Hg between age 4 and 7 months in D2Rj, which decreased to approximately 11 mm Hg in the period from 8 to 10.5 months. In D2J the average IOP showed a steady increase in the observed period from 4 to 10.5 months (from 8.65 to 15.58 mm Hg). Individuals with IOP peaks up to 30 mm Hg were detected in D2Rj, but none of these mice showed signs of an optic neuropathy after 10.5 months. In contrast, 30% of the investigated D2J mice at the age of 10.5 months showed a severe optic neuropathy. Individual data analyses, however, showed no significant correlation between elevated IOP and glaucomatous changes within the D2J population. CONCLUSIONS: Individual correlations of IOP course with axon loss in the single eyes confirmed that in D2J mice, hypertension is not the only causative factor in glaucomatous optic neuropathy. For further investigations on the pathogenesis of glaucoma in D2J mice, the D2Rj strain without a Gpnmb(R150X) mutation and without glaucomatous changes, but with individual IOP elevation, can be used as an interstrain control for D2J.

Enhancement of Outflow Facility in the Murine Eye by Targeting Selected Tight-Junctions of Schlemm's Canal Endothelia.

The juxtacanalicular connective tissue of the trabecular meshwork together with inner wall endothelium of Schlemm's canal (SC) provide the bulk of resistance to aqueous outflow from the anterior chamber. Endothelial cells lining SC elaborate tight junctions (TJs), down-regulation of which may widen paracellular spaces between cells, allowing greater fluid outflow. We observed significant increase in paracellular permeability following siRNA-mediated suppression of TJ transcripts, claudin-11, zonula-occludens-1 (ZO-1) and tricellulin in human SC endothelial monolayers. In mice claudin-11 was not detected, but intracameral injection of siRNAs targeting ZO-1 and tricellulin increased outflow facility significantly. Structural qualitative and quantitative analysis of SC inner wall by transmission electron microscopy revealed significantly more open clefts between endothelial cells treated with targeting, as opposed to non-targeting siRNA. These data substantiate the concept that the continuity of SC endothelium is an important determinant of outflow resistance, and suggest that SC endothelial TJs represent a specific target for enhancement of aqueous movement through the conventional outflow system.

Ultrastructural and biochemical evaluation of the porcine anterior chamber perfusion model.

PURPOSE: To evaluate a porcine anterior chamber perfusion model and to test the transferability of data obtained with this model to the human system. METHODS: Porcine eyes were obtained from a local abattoir and processed within 2 hours after death. Anterior chambers of 42 pairs of eyes were dissected with removal of lens, vitreous, iris, and ciliary processes and perfused for 72 (40 pairs) or 140 (2 pairs) hours with medium or medium supplemented with 10 ng/mL transforming growth factor (TGF)-beta2. Facility was continuously measured. Afterward, trabecular meshwork (TM) specimens from all quadrants were prepared, and sections were analyzed morphologically and with immunohistochemical methods. TM sections of 10 nonperfused pairs of eyes were used as the control. RNA and protein was extracted from the TM specimens. Expression of alphaB-crystallin, fibronectin (FN), plasminogen activator inhibitor (PAI)-1, thrombospondin (TSP)-1, and connective tissue growth factor (CTGF) mRNA and protein in medium-perfused and TGF-beta2-perfused anterior segments was examined by Northern and Western blot analyses. RESULTS: The nonperfused TM showed prominent differences between the temporal and nasal quadrants. Temporally, the ciliary muscle (CM) was pronounced, the scleral sulcus was long and flat, and the scleral spur extended toward the iris root. Nasally, the CM was thin, the sulcus deep, and the spur compact. The outer TM was expanded between the scleral spur and cornea throughout the entire circumference. On the ultrastructural level, the elastic network was connected to the cribriform TM cells and the aqueous plexus endothelium. Perfusion itself had only small effects on the morphology of the outer TM. Aqueous plexus loops remained open, and TM cells showed no signs of necrosis or pyknosis. alphaB-crystallin expression was significantly increased in perfused eyes. Perfusion with TGF-beta2 for 72 hours reduced outflow facility to approximately 60% of that of the medium-perfused control. TM cells adjacent to putative drainage pathways showed enlarged cisterns of rough endoplasmic reticulum (rER), a sign of active protein synthesis. Expression of alphaB-crystallin and FN mRNA were elevated by factors of 5 and 3, respectively. The proteins were upregulated by a factor of 2.5. In addition, TGF-beta2 upregulated PAI-1 (1.7-fold) and TSP-1 (1.6-fold) proteins, two factors shown to be TGF-beta2 responsive in human TM cell culture experiments. CTGF expression was not altered. CONCLUSIONS: These new ultrastructural investigations indicate that the cribriform and subendothelial regions of the porcine TM have an architecture similar to that of the primate TM. The biochemical and physiological response to TGF-beta2 was identical with that described in human TM cell culture and anterior chamber perfusion. The porcine anterior chamber perfusion model is valid for the human system.

Biochemical and morphological analysis of basement membrane component expression in corneoscleral and cribriform human trabecular meshwork cells.

PURPOSE: To determine whether cells of the cribriform trabecular meshwork (TM) express basement membrane (BM) components similar to corneoscleral TM cells and to determine whether cribriform cells are connected to the elastic tendon net of the TM. METHODS: TM cells of the corneoscleral and the cribriform regions were cultured from 10 eyes of 10 donors, aged 20 to 87 years. Cell types were classified by alpha-smooth muscle actin (smA), desmin, and alphaB-crystallin staining. Expression of collagen type IV (ColIV) chains alpha1 to 6; collagen type VIII (ColVIII) alpha1; laminin subunits alpha1 to 5, beta1 to 3, and gamma1 to 3; and nidogen 1 and 2 was tested in both cell types by semiquantitative RT-PCR (sqPCR). Expression of ColIValpha2, ColVIIIalpha1, laminin beta2, and nidogen 1 was quantified by Northern blot analysis. The response to transforming growth factor (TGF)-beta2 treatment was investigated. Serial tangential and sagittal TM sections of 16 eyes from 10 donors (aged 12-90 years) were used for electron- and immunoelectron microscopy. RESULTS: Both TM cell types expressed ColIV chains alpha1, alpha2, alpha4, alpha5, and alpha6; ColVIII alpha1; laminin subunits alpha3, alpha4, beta1, beta2, beta3, gamma1, and gamma2; and nidogen 1, as determined by Northern blot analysis and sqPCR. ColIV alpha3; laminin subunits alpha1, alpha2, and gamma3; and nidogen 2 were not detectable by PCR. Responses to TGF-beta2 treatment did not differ between cell types. In vivo, all cribriform cells were in contact with ColIV containing BM material and were found to connect to the cribriform elastic network. CONCLUSIONS: Cribriform and corneoscleral TM cells show no differences in expression of BM components and response to TGF-beta2. The direct connection of cribriform cells to the elastic tendon network suggests that they are under mechanical tension. This could explain previous findings of alphaB-crystallin expression in the cribriform region.

The prostanoid EP2 receptor agonist butaprost increases uveoscleral outflow in the cynomolgus monkey.

PURPOSE: To investigate the ocular hypotensive effect of the prostanoid EP2 receptor agonist butaprost and to establish its mechanism of action. METHODS: All experiments were performed in cynomolgus monkeys after topical application of butaprost (0.1%). The effects of butaprost on aqueous humor flow were determined by fluorophotometry. Total outflow facility was measured by the two-level, constant-pressure perfusion method, and uveoscleral outflow was determined by perfusion of FITC-labeled dextran through the anterior chamber. Effects on ocular morphology were studied after tissue fixation with transcardial perfusion by paraformaldehyde and immersion fixation of the globe, in animals subjected to long-term treatment with butaprost. Conscious ocular normotensive monkeys and monkeys with unilateral ocular hypertension were used for intraocular pressure (IOP) studies. RESULTS: Butaprost had no significant effect on aqueous humor flow or total outflow facility in ocular normotensive monkeys. Uveoscleral outflow was significantly higher in the butaprost treated eyes than in vehicle treated eyes, 1.03 +/- 0.20 vs. 0.53 +/- 0.18 microL.min(-1). After a 1-year treatment with butaprost, the morphology of the ciliary muscle was changed, showing increased spaces between ciliary muscle bundles and the apparent formation of new outflow channels. In many instances, changes were observed in the trabecular meshwork as well. Butaprost, in a single 0.1% dose, decreased IOP significantly in ocular normotensive monkeys and reduced IOP in laser-induced glaucomatous monkey eyes to the same level as that in the ocular normotensive contralateral eyes. CONCLUSIONS: The prostanoid EP2 receptor agonist butaprost appears to lower IOP by increasing uveoscleral outflow, according to both physiological and morphologic findings. Although the prostanoid EP2 receptor is structurally and functionally distinct from the FP receptor, the effects of EP2 and FP receptor stimulation on aqueous humor outflow are similar.

Histologic findings in pigment dispersion syndrome and pigmentary glaucoma.

PURPOSE: To investigate the morphologic changes in the trabecular meshwork in a case series of eyes with pigment dispersion syndrome and pigmentary glaucoma, and surgical trabeculectomy specimens from eyes with pigmentary glaucoma. MATERIALS AND METHODS: Trabecular meshworks from 6 whole eyes from 3 donors and 7 trabeculectomy specimens were studied by light and electron microscopy. Axonal counts from the whole eyes were correlated with qualitative and quantitative data of meshwork changes. RESULTS: Changes in the meshwork varied around the circumference of the eyes, but in all 6 eyes in most regions of the circumference there were numerous pigment granules within trabecular cells; pigment was not found within intertrabecular or cribriform spaces. In some regions of the circumference there was trabecular cell loss, loss of intertrabecular spaces, fusion of lamellae, and an increase in extracellular material under the inner wall of the canal. Separation of the normal tendinous connection to the canal wall cells was noted in some regions of all eyes. This change could be associated with regions of pathologic separation of the inner wall from the cribriform region, associated with partial obliteration of the lumen of the canal with cells and cell processes. In eyes with pronounced axon loss, meshworks showed most pronounced loss of trabecular cells and increased extracellular material. Trabeculectomy specimens had similar changes and, in addition, showed damaged trabecular cells and collapse of intertrabecular spaces without fusion of lamellae, consistent with artifacts from manipulation during surgery. CONCLUSIONS: Loss of trabecular cells, fusion of trabecular lamellae with collapse of intertrabecular spaces, increase in extracellular material, and obliteration of the canal were found in various amounts around the circumference of eyes with pigment dispersion syndrome and elevated intraocular pressure, and pigmentary glaucoma. These probably all contribute to the development of increased intraocular pressure. Meshworks from trabeculectomy specimens showed these findings and also showed artifactual damage of cells and loss of intertrabecular spaces. This suggests that handling during surgery may cause single trabeculectomy specimens to give only an incomplete picture of the pathophysiology of pigmentary glaucoma.

Pathophysiologic changes in the optic nerves of eyes with primary open angle and pseudoexfoliation glaucoma.

PURPOSE: To determine whether differences in the optic nerve occur in eyes with primary versus secondary open-angle glaucoma. METHODS: Optic nerves obtained at autopsy from 36 eyes with primary open-angle glaucoma (POAG) and 15 with pseudoexfoliation glaucoma (PEXG) were studied quantitatively and qualitatively. Axon counts, fibrosis, capillary number and density, and arteriosclerotic changes were assessed in the postlaminar optic nerve and compared to normal age-matched autopsy eyes. Changes in composition of extracellular matrix components were evaluated by immunohistochemistry and electron microscopy. RESULTS: Marked differences were found between POAG and PEXG. Axon loss in eyes with POAG but not in PEXG was associated with increasing connective tissue in the septa and surrounding the central retinal vessels, including increased amounts of type IV and VI collagen. The total number of capillaries decreased with the loss of axons in both POAG and PEXG. POAG nerves, however, had a decrease in the density of capillaries, whereas in PEXG the capillary density did not change with axon loss. Arteriosclerotic changes were more common in glaucomatous eyes than in age-matched control eyes. CONCLUSIONS: The difference in morphology of the optic nerves between POAG and PEXG indicates that in eyes with POAG, elevated IOP cannot be the only pathogenetic factor in glaucomatous optic neuropathy. Additional factors, inducing fibrosis and loss of capillaries, seem to be involved. Such additional factors may also contribute to the clinical finding in POAG that nerves can become damaged without elevation of intraocular pressure.