Segmental differences found in aqueous angiographic-determined high - and low-flow regions of human trabecular meshwork.

This work sought to compare aqueous angiographic segmental patterns with bead-based methods which directly visualize segmental trabecular meshwork (TM) tracer trapping. Additionally, segmental protein expression differences between aqueous angiographic-derived low- and high-outflow human TM regions were evaluated. Post-mortem human eyes (One Legacy and San Diego eye banks; n = 15) were perfused with fluorescent tracers (fluorescein [2.5%], indocyanine green [0.4%], and/or fluorescent microspheres). After angiographic imaging (Spectralis HRA+OCT; Heidelberg Engineering), peri-limbal low- and high-angiographic flow regions were marked. Aqueous angiographic segmental outflow patterns were similar to fluorescent microsphere TM trapping segmental patterns. TM was dissected from low- and high-flow areas and processed for immunofluorescence or Western blot and compared. Versican expression was relatively elevated in low-flow regions while MMP3 and collagen VI were relatively elevated in high-flow regions. TGF-ß2, thrombospondin-1, TGF-ß receptor1, and TGF-ß downstream proteins such as α-smooth muscle actin were relatively elevated in low-flow regions. Additionally, fibronectin (FN) levels were unchanged, but the EDA isoform (FN-EDA) that is associated with fibrosis was relatively elevated in low-flow regions. These results show that segmental aqueous angiographic patterns are reflective of underlying TM molecular characteristics and demonstrate increased pro-fibrotic activation in low-flow regions. Thus, we provide evidence that aqueous angiography outflow visualization, the only tracer outflow imaging method available to clinicians, is in part representative of TM biology.

Micropulse Transscleral Diode Laser Cyclophotocoagulation in Refractory Glaucoma: Short-Term Efficacy, Safety, and Impact of Surgical History on Outcomes.

PURPOSE: To assess the short-term efficacy and safety of micropulse transscleral diode laser cyclophotocoagulation (MP-TSCPC) in the management of refractory glaucoma and to compare outcomes based on prior glaucoma surgeries. DESIGN: Retrospective analysis. PARTICIPANTS: Patients with refractory glaucoma who underwent MP-TSCPC at a single institution by 1 of 4 surgeons. METHODS: Chart review of cases of MP-TSCPC using the Iridex Cyclo G6 (Mountain View, CA) laser with standard parameters and laser duration at the discretion of each treating physician. MAIN OUTCOME MEASURES: Probability of postoperative success was estimated by the Kaplan-Meier method. Success parameters included intraocular pressure (IOP) 6 to 21 mmHg with or without topical antihypertensive therapy, 20% or more IOP reduction from baseline for any 2 consecutive visits after 3 postoperative months, and no subsequent glaucoma surgery. RESULTS: One hundred sixteen eyes of 116 patients (mean age, 65.8±16.9 years) were included. Baseline IOP was 22.2±7.9 mmHg, and mean postoperative follow-up time was 6.3±3.4 months (range, 3-12 months.) Postoperative IOP at the final follow up was 15.3±6.6 mmHg (P < 0.01), corresponding to a reduction of approximately 6.9 mmHg (31.1%). Most eyes (66.4%) underwent at least 6 months of follow-up. Short-term probability of success was 93.1% at 3 months and 74.3% at 6 months. Eyes that had undergone prior traditional glaucoma surgery (trabeculectomy, tube shunt, excessive pressure-regulating shunt system miniature glaucoma shunt [Alcon, Fort Worth, TX], or a combination thereof) demonstrated a higher probability of success (67.6%) compared with eyes that had not (41.4%; P = 0.014). The most common complications were decline in best-corrected visual acuity (7.8%) and hypotony (1.7%). CONCLUSIONS: Micropulse transscleral diode laser cyclophotocoagulation has a significant short-term ocular hypotensive effect and favorable safety profile in eyes with refractory glaucoma. The probability of successful outcome was greater in eyes that had undergone prior traditional glaucoma surgery.

Deep tissue analysis of distal aqueous drainage structures and contractile features.

Outflow resistance in the aqueous drainage tract distal to trabecular meshwork is potentially an important determinant of intraocular pressure and success of trabecular bypass glaucoma surgeries. It is unclear how distal resistance is modulated. We sought to establish: (a) multimodal 2-photon deep tissue imaging and 3-dimensional analysis of the distal aqueous drainage tract (DT) in transgenic mice in vivo and ex vivo; (b) criteria for distinguishing the DT from blood and lymphatic vessels; and (c) presence of a DT wall organization capable of contractility. DT lumen appeared as scleral collagen second harmonic generation signal voids that could be traced back to Schlemm's canal. DT endothelium was Prox1-positive, CD31-positive and LYVE-1-negative, bearing a different molecular signature from blood and true lymphatic vessels. DT walls showed prominent filamentous actin (F-actin) labeling reflecting cells in a contracted state. F-actin co-localized with mesenchymal smooth muscle epitopes of alpha-smooth muscle actin, caldesmon and calponin, which localized adjacent and external to the endothelium. Our findings support a DT wall organization resembling that of blood vessels. This reflects a capacity to contract and support dynamic alteration of DT caliber and resistance analogous to the role of blood vessel tone in regulating blood flow.

Sustained Resolution of Macular Retinoschisis After Trabeculectomy in a Patient With Progressive Glaucoma.

PURPOSE: To evaluate the clinical characteristics of a patient with primary open-angle glaucoma in whom macular retinoschisis resolved completely after trabeculectomy consistently lowered intraocular pressure (IOP). METHODS: A single case report. RESULTS: We report a case of retinoschisis involving the macula in a patient with primary open-angle glaucoma in the absence of myopic maculopathy, optic nerve anomaly, or x-linked retinoschisis. The patient's glaucoma was associated with progressive visual field loss in the setting of IOP fluctuations related to posture. A trabeculectomy reduced IOP and posture-related IOP fluctuations with subsequent resolution of macular retinoschisis. In the 1-year postoperative period following trabeculectomy, the patient has remained without retinoschisis and visual fields have been stable. CONCLUSIONS: Improved IOP control resulting in resolution of retinoschisis may distinguish retinoschisis associated with glaucoma from other forms of retinoschisis.

Toward in vivo two-photon analysis of mouse aqueous outflow structure and function.

The promise of revolutionary insights into intraocular pressure (IOP) and aqueous humor outflow homeostasis, IOP pathogenesis, and novel therapy offered by engineered mouse models has been hindered by a lack of appropriate tools for studying the aqueous drainage tissues in their original 3-dimensional (3D) environment. Advances in 2-photon excitation fluorescence imaging (TPEF) combined with availability of modalities such as transgenic reporter mice and intravital dyes have placed us on the cusp of unlocking the potential of the mouse model for unearthing insights into aqueous drainage structure and function. Multimodality 2-photon imaging permits high-resolution visualization not only of tissue structural organization but also cells and cellular function. It is possible to dig deeper into understanding the cellular basis of aqueous outflow regulation as the technique integrates analysis of tissue structure, cell biology and physiology in a way that could also lead to fresh insights into human glaucoma. We outline recent novel applications of two-photon imaging to analyze the mouse conventional drainage system in vivo or in whole tissues: (1) collagen second harmonic generation (SHG) identifies the locations of episcleral vessels, intrascleral plexuses, collector channels, and Schlemm's canal in the distal aqueous drainage tract; (2) the prospero homeobox protein 1-green fluorescent protein (GFP) reporter helps locate the inner wall of Schlemm's canal; (3) Calcein AM, siGLO™, the fluorescent reporters m-Tomato and GFP, and coherent anti-Stokes scattering (CARS), are adjuncts to TPEF to identify live cells by their membrane or cytosolic locations; (4) autofluorescence and sulforhodamine-B to identify elastic fibers in the living eye. These tools greatly expand our options for analyzing physiological and pathological processes in the aqueous drainage tissues of live mice as a model of the analogous human system.

Pilot Study of Lamina Cribrosa Intensity Measurements in Glaucoma Using Swept-Source Optical Coherence Tomography.

PURPOSE: To compare the lamina cribrosa (LC) intensity in glaucoma-suspect eyes and eyes with mild to moderate glaucoma using swept-source optical coherence tomography. METHODS: Optic disc volume scans were collected using swept-source optical coherence tomography in 19 clinically defined glaucoma-suspect eyes and 29 eyes with mild to moderate glaucoma. LC intensity was measured using Image J software, and the resultant values were normalized using the retinal pigment epithelium and vitreous signal. RESULTS: Mean age was 53.7±18.5 years in the glaucoma-suspect eyes and 63.0±16.1 years in the eyes with mild to moderate glaucoma (P=0.161). Significant differences in LC intensity were observed between the 2 groups, with median LC intensity values of 0.96 and 0.86 units in the glaucoma-suspect and the mild to moderate glaucoma groups, respectively (P<0.001). A weak positive correlation was found between mean deviation and normalized LC intensity (r=0.344; P=0.018). CONCLUSIONS: Intensity measurement of the LC is a potential novel parameter which warrants further study in the setting of glaucoma.

Aqueous Angiography with Fluorescein and Indocyanine Green in Bovine Eyes.

PURPOSE: We characterize aqueous angiography as a real-time aqueous humor outflow imaging (AHO) modality in cow eyes with two tracers of different molecular characteristics. METHODS: Cow enucleated eyes (n = 31) were obtained and perfused with balanced salt solution via a Lewicky AC maintainer through a 1-mm side-port. Fluorescein (2.5%) or indocyanine green (ICG; 0.4%) were introduced intracamerally at 10 mm Hg individually or sequentially. With an angiographer, infrared and fluorescent images were acquired. Concurrent anterior segment optical coherence tomography (OCT) was performed, and fixable fluorescent dextrans were introduced into the eye for histologic analysis of angiographically positive and negative areas. RESULTS: Aqueous angiography in cow eyes with fluorescein and ICG yielded high-quality images with segmental patterns. Over time, ICG maintained a better intraluminal presence. Angiographically positive, but not negative, areas demonstrated intrascleral lumens with anterior segment OCT. Aqueous angiography with fluorescent dextrans led to their trapping in AHO pathways. Sequential aqueous angiography with ICG followed by fluorescein in cow eyes demonstrated similar patterns. CONCLUSIONS: Aqueous angiography in model cow eyes demonstrated segmental angiographic outflow patterns with either fluorescein or ICG as a tracer. TRANSLATIONAL RELEVANCE: Further characterization of segmental AHO with aqueous angiography may allow for intelligent placement of trabecular bypass minimally invasive glaucoma surgeries for improved surgical results.

Aqueous Angiography-Mediated Guidance of Trabecular Bypass Improves Angiographic Outflow in Human Enucleated Eyes.

PURPOSE: To assess the ability of trabecular micro-bypass stents to improve aqueous humor outflow (AHO) in regions initially devoid of AHO as assessed by aqueous angiography. METHODS: Enucleated human eyes (14 total from 7 males and 3 females [ages 52-84]) were obtained from an eye bank within 48 hours of death. Eyes were oriented by inferior oblique insertion, and aqueous angiography was performed with indocyanine green (ICG; 0.4%) or fluorescein (2.5%) at 10 mm Hg. With an angiographer, infrared and fluorescent images were acquired. Concurrent anterior segment optical coherence tomography (OCT) was performed, and fixable fluorescent dextrans were introduced into the eye for histologic analysis of angiographically positive and negative areas. Experimentally, some eyes (n = 11) first received ICG aqueous angiography to determine angiographic patterns. These eyes then underwent trabecular micro-bypass sham or stent placement in regions initially devoid of angiographic signal. This was followed by fluorescein aqueous angiography to query the effects. RESULTS: Aqueous angiography in human eyes yielded high-quality images with segmental patterns. Distally, angiographically positive but not negative areas demonstrated intrascleral lumens on OCT images. Aqueous angiography with fluorescent dextrans led to their trapping in AHO pathways. Trabecular bypass but not sham in regions initially devoid of ICG aqueous angiography led to increased aqueous angiography as assessed by fluorescein (P = 0.043). CONCLUSIONS: Using sequential aqueous angiography in an enucleated human eye model system, regions initially without angiographic flow or signal could be recruited for AHO using a trabecular bypass stent.

Optimizing two-photon multiple fluorophore imaging of the human trabecular meshwork.

PURPOSE: Advances in two-photon (2P) deep tissue imaging provide powerful options for simultaneously viewing multiple fluorophores within tissues. We determined imaging parameters for optimally visualizing three fluorophores in the human trabecular meshwork (TM) to simultaneously detect broad-spectrum autofluorescence and multiple fluorophores through a limited number of emission filters. METHODS: 2P imaging of viable human postmortem TM was conducted to detect Hoechst 33342-labeled nuclei, Alexa-568-conjugated phalloidin labeling of filamentous actin, and autofluorescence of the structural extracellular matrix (ECM). Emission detection through green (500-550 nm), near-red (565-605 nm), and far-red (590-680 nm) filters following 2P excitation at 750, 800, 850, and 900 nm was analyzed. Region-of-interest (ROI) image analysis provided fluorescence intensity values for each fluorophore. RESULTS: Red-channel Alexa 568 fluorescence was of highest intensity with 2P 750 nm and 800 nm excitation. Alexa 568 was imperceptible with 900 nm excitation. With excitation at 750 nm and 800 nm, Hoechst 33,342 intensity swamped autofluorescence in the green channel, and marked bleed-through into red channels was seen. 850 nm excitation yielded balanced Hoechst 33342 and autofluorescence intensities, minimized their bleed-through into the far-red channel, and produced reasonable Alexa 568 intensities in the far-red channel. CONCLUSIONS: 2P excitation at 850 nm and long-wavelength emission detection in the far-red channel allowed simultaneous visualization of the specific mix of endogenous and exogenous fluorophores with reasonably balanced intensities while minimizing bleed-through when imaging the human TM.

Tissue-based multiphoton analysis of actomyosin and structural responses in human trabecular meshwork.

The contractile trabecular meshwork (TM) modulates aqueous humor outflow resistance and intraocular pressure. The primary goal was to visualize and quantify human TM contractile state by analyzing actin polymerization (F-actin) by 2-photon excitation fluorescence imaging (TPEF) in situ. A secondary goal was to ascertain if structural extracellular matrix (ECM) configuration changed with contractility. Viable ex vivo human TM was incubated with latrunculin-A (Lat-A) or vehicle prior to Alexa-568-phalloidin labeling and TPEF. Quantitative image analysis was applied to 2-dimensional (2D) optical sections and 3D image reconstructions. After Lat-A exposure, (a) the F-actin network reorganized as aggregates; (b) F-actin-associated fluorescence intensity was reduced by 48.6% (mean; p = 0.007; n = 8); (c) F-actin 3D distribution was reduced by 68.9% (p = 0.040); (d) ECM pore cross-sectional area and volume were larger by 36% (p = 0.032) and 65% (p = 0.059) respectively and pores appeared more interconnected; (e) expression of type I collagen and elastin, key TM structural ECM proteins, were unaltered (p = 0.54); and (f) tissue viability was unchanged (p = 0.39) relative to vehicle controls. Thus Lat-A-induced reduction of actomyosin contractility was associated with TM porous expansion without evidence of reduced structural ECM protein expression or cellular viability. These important subcellular-level dynamics could be visualized and quantified within human tissue by TPEF.

Aqueous Angiography: Real-Time and Physiologic Aqueous Humor Outflow Imaging.

PURPOSE: Trabecular meshwork (TM) bypass surgeries attempt to enhance aqueous humor outflow (AHO) to lower intraocular pressure (IOP). While TM bypass results are promising, inconsistent success is seen. One hypothesis for this variability rests upon segmental (non-360 degrees uniform) AHO. We describe aqueous angiography as a real-time and physiologic AHO imaging technique in model eyes as a way to simulate live AHO imaging. METHODS: Pig (n = 46) and human (n = 6) enucleated eyes were obtained, orientated based upon inferior oblique insertion, and pre-perfused with balanced salt solution via a Lewicky AC maintainer through a 1mm side-port. Fluorescein (2.5%) was introduced intracamerally at 10 or 30 mm Hg. With an angiographer, infrared and fluorescent (486 nm) images were acquired. Image processing allowed for collection of pixel information based on intensity or location for statistical analyses. Concurrent OCT was performed, and fixable fluorescent dextrans were introduced into the eye for histological analysis of angiographically active areas. RESULTS: Aqueous angiography yielded high quality images with segmental patterns (p<0.0001; Kruskal-Wallis test). No single quadrant was consistently identified as the primary quadrant of angiographic signal (p = 0.06-0.86; Kruskal-Wallis test). Regions of high proximal signal did not necessarily correlate with regions of high distal signal. Angiographically positive but not negative areas demonstrated intrascleral lumens on OCT images. Aqueous angiography with fluorescent dextrans led to their trapping in AHO pathways. CONCLUSIONS: Aqueous angiography is a real-time and physiologic AHO imaging technique in model eyes.

Endoscopic Cyclophotocoagulation and Pars Plana Ablation (ECP-plus) to Treat Refractory Glaucoma.

PURPOSE: To report clinical outcomes after pars plana endoscopic cyclophotocoagulation of the ciliary processes and pars plana (ECP-plus), a novel treatment for refractory glaucoma. DESIGN: Retrospective, noncomparative interventional case series. SETTING: multicenter tertiary referral academic and clinical practice. STUDY POPULATION: fifty-three eyes of 53 consecutive subjects undergoing ECP-plus who had uncontrolled intraocular pressure (IOP) despite prior glaucoma surgeries and maximally tolerated medical therapy. OBSERVATION PROCEDURE: retrospective analysis of clinical data after ECP-plus and pars plana vitrectomy. MAIN OUTCOME MEASURES: primary outcome was IOP at 6 and 12 months. Secondary outcomes were number of glaucoma medications and postoperative complications. RESULTS: Diagnoses were primary open-angle glaucoma (32%), chronic angle-closure glaucoma (26%), and secondary open-angle glaucoma (OAG, 42%); 50/53 of subjects had 6 months' and 28/53 had 12 months' follow-up data. Preoperative IOP was 27.9±7.5 mm Hg (mean±SD). Postoperative IOP at 6 mo was 10.2±5.6 and at 12 mo was 10.7±5.2 lower than preoperative levels (all P<0.0001). Cumulative treatment success was 81% at 6 mo and 78% at 12 mo. Number of medications fell from 3.4±1.2 pretreatment to 0.8±1.0 at 1 to 6 mo and 0.7±1.2 at 12 mo postoperatively (all P<0.0001). Complications in the initial postoperative period (<3 mo) were hypotony (3/53, 6%), fibrinous uveitis (2/53, 4%), and cystoid macular edema without hypotony (CME; 4/53, 6%). Complications beyond 6 mo occurred in 8/50 (16%) subjects as hypotony (4/50, 8%), choroidal detachment (4/50, 3 with IOP<5 and 1 with IOP≥5; 8%), CME without hypotony (3/50, 6%), and failed corneal graft (1/50, 2%). CONCLUSIONS: The mean IOP was lowered by at least 61% after ECP-plus and IOP lowering was sustained over the follow-up period. Fewer glaucoma medications were needed. Complication rates were comparable with or slightly higher than anterior endoscopic cyclophotocoagulation and acceptable given the refractory nature of disease being treated.

Feedback-controlled constant-pressure anterior chamber perfusion in live mice.

PURPOSE: To describe live mouse, anterior chamber constant-pressure perfusion by an approach using feedback-controlled coupling of pressure and flow to maintain a preset pressure. METHODS: We established a microperfusion system that maintains a constant preset pressure in the anterior chamber of live mice by automatically regulating the microsyringe pump flow rate with a computer-controlled voltage feedback loop. Perfusion was by single-needle cannulation. We characterized the following in C57BL/6 mice aged 3-4 months in vivo: (i) pressure stability, (ii) pressure and flow rate reproducibility, (iii) total outflow facility, and (iv) anterior segment histology after perfusion. RESULTS: Twenty live mice underwent perfusion. Constant pressure was quickly attained and stably maintained. The coefficient of pressure variation over time during perfusion at a preset pressure was <0.001. The average coefficient of variation for repeat pressure and flow rate measurements was 0.0005 and 0.127, respectively. The relationship between flow rate and pressure was linear for perfusions between 15 and 35 mmHg. The total outflow facility was 0.0066 µl/min/mmHg. Perfusion system resistance (0.5 mmHg/min/µl) was negligible relative to the ocular outflow resistance (147 mmHg/min/µl) at physiologically relevant perfusion pressures of 15-35 mmHg. No histological disruption of the drainage tissue was seen following perfusion. CONCLUSIONS: Predetermined pressure was stably maintained during constant-pressure perfusion of live mouse eyes by a method using feedback-controlled coupling of pressure and flow along with single-needle anterior chamber cannulation. Perfusion measurements were reproducible. This approach is potentially useful for exploring aqueous drainage tissue biology, physiology, and pharmacology in live mice.

Tissue-based imaging model of human trabecular meshwork.

We have developed a tissue-based model of the human trabecular meshwork (TM) using viable postmortem corneoscleral donor tissue. Two-photon microscopy is used to optically section and image deep in the tissue to analyze cells and extracellular matrix (ECM) within the original three-dimensional (3D) environment of the TM. Multimodal techniques, including autofluorescence (AF), second harmonic generation (SHG), intravital dye fluorescence, and epifluorescence, are combined to provide unique views of the tissue at the cellular and subcellular level. SHG and AF imaging are non-invasive tissue imaging techniques with potential for clinical application, which can be modeled in the system. We describe the following in the tissue-based model: analysis of live cellularity to determine tissue viability; characteristics of live cells based on intravital labeling; features and composition of the TM's structural ECM; localization of specific ECM proteins to regions such as basement membrane; in situ induction and expression of tissue markers characteristic of cultured TM cells relevant to glaucoma; analysis of TM actin and pharmacological effects; in situ visualization of TM, inner wall endothelium, and Schlemm's canal; and application of 3D reconstruction, modeling, and quantitative analysis to the TM. The human model represents a cost-effective use of valuable and scarce yet available human tissue that allows unique cell biology, pharmacology, and translational studies of the TM.

Contractile markers distinguish structures of the mouse aqueous drainage tract.

PURPOSE: Structures of the aqueous humor drainage tract are contractile, although the tract is not entirely composed of muscle. We characterized the mouse aqueous drainage tract by immunolabeling contractile markers and determined whether profiling these markers within the tract distinguished its key structures of the trabecular meshwork (TM) and ciliary muscle (CM). METHODS: Enucleated eyes from pigmented C57BL/6 (n=8 mice) and albino BALB/c (n=6 mice) mice were processed for cryo- and formalin-fixed paraffin-embedded sectioning. Immunofluorescence labeling was performed for the following: (a) filamentous actin (using fluorescence-conjugated phalloidin), representing a global contractile marker; (b) α-smooth muscle actin (α-SMA), caldesmon, and calponin, representing classic smooth muscle epitopes; and (c) nonmuscle myosin heavy chain, representing a nonmuscle contractile protein. Tissue labeling was identified by confocal microscopy and analyzed quantitatively. Hematoxylin and eosin staining provided structural orientation. RESULTS: A small portion of the TM faced the anterior chamber; the rest extended posteriorly alongside Schlemm's canal (SC) within the inner sclera. Within the drainage tract, filamentous actin labeling was positive in TM and CM. α-SMA and caldesmon labeling was seen primarily along the CM, which extended from the anterior chamber angle to its posterior termination beyond the SC near the retina. Low intensity, patchy α-SMA and caldesmon labeling was seen in the TM. Myosin heavy chain immunoreactivity was primarily found in the TM and calponin was primarily observed in the CM. C57BL/6 and BALB/c comparison showed that pigment obscured fluorescence in the ciliary body. CONCLUSIONS: Our strategy of profiling contractile markers distinguished mouse aqueous drainage tract structures that were otherwise indistinguishable by hematoxylin and eosin staining. The mouse TM was seen as an intervening structure between SC, a part of the conventional drainage tract, and CM, a part of the unconventional drainage tract. Our findings provide important insights into the structural and functional organization of the mouse aqueous drainage tract and a basis for exploring the role of contractility in modulating aqueous outflow.

Sources of structural autofluorescence in the human trabecular meshwork.

PURPOSE: In situ 2-photon excitation fluorescence (TPEF) of the human trabecular meshwork (TM) reveals beams of heterogeneous autofluorescence (AF) comprising high intensity fluorescent fibers (AF-high) on a background of lower intensity fluorescence (AF-low). To determine the sources of this AF heterogeneity, we imaged human TM to characterize AF, second harmonic generation (SHG) for collagen, and eosin-labeled fluorescence identifying elastin. METHODS: Corneoscleral rims retained after corneal transplantation were incubated with and without eosin, and imaged by TPEF. TPEF was collected through multiphoton bandpass filters to obtain AF, SHG (collagen bandwidth), and eosin-labeled fluorescence images. For qualitative comparisons, near-simultaneous image acquisition pairs of AF-SHG (+/-eosin coincubation), AF-eosin, and SHG-eosin were captured. For quantitative comparisons, multiple regions of interest (ROI) were defined in separate TM beam regions within the uveal and corneoscleral meshwork for image acquisition pairs of AF-SHG (without eosin coincubation) and SHG-eosin. We defined 18 ROI within each acquisition pair as the basis for Manders colocalization analysis. Perfect colocalization was defined as a Manders coefficient (Mcoeff) of 1. RESULTS: Qualitatively and quantitatively, AF-low colocalized with SHG (Mcoeff=1), but not SHG signal-voids. AF-high colocalized with SHG signal-voids (Mcoeff=1), but not the SHG signal. Like AF-high, eosin-labeled fluorescence qualitatively and quantitatively colocalized (Mcoeff=1) with SHG signal-voids, but not the SHG signal. CONCLUSIONS: Heterogeneous AF in human TM is comprised of high intensity signal originating from elastin fibers in beam cores and lower intensity signal originating from collagen. These findings are relevant to interpreting structural extracellular matrix signals in AF images of the TM.

Analyzing live cellularity in the human trabecular meshwork.

PURPOSE: To directly visualize the live cellularity of the intact human trabecular meshwork (TM) and quantitatively analyze tissue viability in situ. METHODS: Human donor corneoscleral rims were sectioned immediately before intravital dye incubation to label nuclei (Hoechst 33342 & propidium iodide [PI]); cytosol (CellTracker Red CMTPX, calcein AM); and membranes (octadecyl rhodamine B chloride [R18]), followed by 2-photon microscopy. Viability was assessed by counting cells in tissue colabeled with PI and Calcein AM. Some tissues were exposed to Triton X-100 to establish dead tissue controls. Fresh postmortem eyes (within 48 hours of death) represented viable tissue controls. Tissues with live cellularity exceeding 50% were considered viable. RESULTS: Hoechst nuclear labeling was seen throughout the TM, among the autofluorescent beams, plate-like structures and fibers of the meshwork, and within tissue gaps and pores. CellTracker-labeled live cells were attached to autofluorescent TM structures and filled corneoscleral meshwork pores. R18-labeling revealed the membrane distributions of interconnected cells. Calcein-positive cells were visible in all TM layers, but not in tissues killed by Triton X-100 exposure. Dead control tissues showed PI staining in the absence of Calcein-positive cells. Two-thirds of the standard donor tissues we received possessed viable TM, having a mean live cellularity of 71% (n = 14), comparable with freshly postmortem eyes (76%; n = 2). Mean live cellularity of nonviable tissue was 11% (n = 7). CONCLUSIONS: We have visualized and quantified the live cellularity of the TM in situ. This provided unique perspectives of live cell-matrix organization and a means of assaying tissue viability.

Two-photon immunofluorescence characterization of the trabecular meshwork in situ.

PURPOSE: To develop an in situ model to study biological responses and glaucoma pathology in the human trabecular meshwork (TM). Characteristic TM cell- and glaucoma-associated markers were localized in situ in relation to the tissue's autofluorescent structural extracellular matrix (ECM) by two-photon excitation fluorescence optical sectioning (TPEF). METHODS: Human donor corneoscleral (CS) tissue containing the intact aqueous drainage tract was incubated with dexamethasone (Dex) or TGF-ß1, and immunostained for epifluorescence (EF) microscopy with antibodies to myocilin and alpha smooth muscle (α-SMA). Separate specimens were labeled for Type-IV collagen and fibronectin. Nuclei were stained with Hoechst 33342. Multimodal TPEF was used to visualize EF, intravital dyes, and autofluorescence (AF) in situ. Three-dimensional (3D) localization of fluorescence within the TM was analyzed using reconstruction software. RESULTS: Autofluorescent beams, perforated sheets, and fibers, consistent with the uveal (UV), CS, and juxtacanalicular (JCT) meshwork, respectively, were captured at different depths of the TM. Type-IV collagen EF distinctly outlined the AF beams in a location consistent with basement membrane. Fibronectin EF showed a diffuse reticular pattern throughout the TM. TGF-ß1-induced α-SMA expression, which was distributed perinuclearly in cells among autofluorescent structures. Dex-induced myocilin expression had both cytosolic and extracellular distributions. CONCLUSIONS: The authors have localized markers that are characteristic of TM cells and relevant to glaucoma pathogenesis in situ using multimodal TPEF without conventional histological embedding and sectioning. Protein expression was inducible in situ and could be analyzed with respect to cells and the ECM within the 3D environment of the human TM.