2.1 Culture and characterization of human TM cells
Great care should be taken in obtaining and characterizing human TM cells (please see [25] and [26] for details). Human TM cell culture generally occurs through the careful dissection of TM tissue from human donor eyes. The human donor eyes should be obtained from a reputable source that follows the tenets of the Declaration of Helsinki with donor or family consent. TM cells are grown from TM tissue explants [19, 27] or from cells proteolytically isolated from the TM tissue prior to culture [28]. Although culture conditions vary between laboratories, TM cells are usually cultured in DMEM or a DMEM/hybrid medium that contains L-glutamine, antibiotics, and fetal bovine serum (10-20%), with medium changes 2-3 times per week. We have found that the TM cells grow better when some of the conditioned medium is left in culture (~10-20%) rather than totally replacing the conditioned medium with fresh medium. Although there is no universal marker that differentiates TM cells from other cell types, the consensus recommendation [25, 26] is the TM cells should be authenticated by a combination of: (1) morphology (cells should grow as confluent monolayers with overlapping boundaries), (2) dexamethasone (DEX)-induction of the glaucoma gene Myocilin (MYOC), and (3) a combination of other markers such as a-smooth muscle actin, collagens I, II, & IV, and laminin. Examination of CLANs should be conducted in confluent TM cells.
2.2 Experimental induction of CLANs in confluent TM cells
Although TM cells have high propensity to form CLANs compared to other cell types in the anterior segment, the level of background CLANs accounts is only 4% in normal human TM cells at confluency [27] [29]. The structure of CLANs can vary greatly from a single CLAN with a central “hub” connected to 5-6 “spokes” (Figure 2A) to very extensive cross-linked actin networks (Figure 3A). This low prevalence of CLANs makes it difficult to study CLANs-associated pathology in normal TM cells. The prevalence of CLANs increases to 25-40% in glaucomatous human TM cells [19] (Figures 3A, D). However, cells and tissues derived from glaucoma donors are less commonly available. We have shown that glaucoma-inducing agents like the glucocorticoid dexamethasone (DEX) [24, 27, 29, 30] (Figures 3C-D) and transforming growth factor b-2 (TGFb-2) [31, 32] (Figures 2A-B) can induce CLAN formation in normal TM cells [32]. Experimental induction of CLANs using DEX or TGFb2 is now commonly used to study the role of CLANs in glaucoma pathology and to test new glaucoma targets. Given the relevance of CLANs to glaucoma, determining the molecular mechanisms of glaucoma pathophysiology may lead to the discovery of new glaucoma disease modifying therapies.
2.2.1 Glucocorticoid (GC) induction of CLANS (Figures 3C-D)
Glucocorticoids (GCs) are a widely prescribed class of synthetic steroids known for their potent anti-inflammatory and immunomodulatory effects. GCs remain the gold standard in treating ocular inflammation associated with disease or surgery. Unfortunately, prolonged GC treatment has been associated with numerous side effects including GC-induced ocular hypertension (GC-OHT) that can lead to iatrogenic secondary open-angle glaucoma [33]. Since the clinical and molecular manifestations of GC-OHT resemble POAG, potent GCs such as dexamethasone (DEX) have been increasingly used to experimentally probe POAG pathology [29, 34]. DEX treatment increases ECM deposition and induces cytoskeletal reorganization in TM cells causing cell stiffening, which is also observed in cells from POAG patients [7, 19, 20]. We have previously shown that DEX treatment of cultured normal human TM cells can induce CLAN formation [24, 27] (Figure 2C-D). This experimental induction of CLANs in now widely used to study CLANs biology and determine its pathological relevance to glaucoma.
Here we describe induction of CLANs in normal human TM cells isolated from non-glaucomatous donor eyes as described previously [24, 27].
- Culture and characterization of human TM cells is described in section 2.1
- Primary human TM cells are grown and maintained in Dulbecco’s modified Eagle’s medium (DMEM)–low glucose (Invitrogen-Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Atlas Biologicals, Fort Collins, CO, USA), penicillin (100 units/mL), streptomycin (0.1 mg/mL), and L-glutamine (0.292 mg/mL) (Thermo Fisher Scientific).
- For experiments requiring microscopy, cells are plated on round glass coverslips placed in 24-well plates or on glass-chambered slides.
- Prepare and filter the stock DEX solution (10 µM dexamethasone in ethanol) and vehicle solution (ethanol) using a 0.22 µM syringe filter. Culture the NTM cells until confluent and then treat with vehicle control (0.1% ethanol) or DEX (100nM in 0.1% ethanol) added to culture medium for another 7-14 days, replacing with fresh medium 2 times per week.
2.2.2 Transforming growth factor b2 (TGFb2) mediated induction of CLANs (Figure 2A-B)
The profibrotic cytokine transforming growth factor b2 (TGFb2) is elevated in aqueous humor [35-37] and TM cells [38] derived from POAG patients and has been associated with glaucomatous damage to the TM [36, 37] [39]. In addition, TGFb2 causes ocular hypertension in perfusion cultured human anterior segments [39, 40] and in mice [41, 42]. Furthermore, aqueous humor by itself is also known to induce CLANs formation [31] presumably due to the TGFb2 in aqueous humor. We previously have shown that both SMAD-dependent and SMAD-independent TGFb2 signaling contribute to actin cytoskeleton reorganization and induction of CLANs [32].
- For TGFb2 mediated induction of CLANs, confluent cultured TM cells should be cultured in low serum conditions (0.25-1.0% FBS) to reduce confounding effects from growth factors present in the serum. To enhance cell survival in low serum conditions, use growth medium containing high glucose.
- Culture the confluent TM cells in DMEM–high glucose (Invitrogen-Gibco Life Technologies, Grand Island, NY, USA) supplemented with 0.25-1 % fetal bovine serum (Atlas Biologicals, Fort Collins, CO, USA), penicillin (100 units/mL), streptomycin (0.1 mg/mL), and L-glutamine (0.292 mg/mL) (Thermo Fisher Scientific).
- For experiments requiring microscopy, plate cells on round glass coverslips placed in 24-well plates or on glass-chambered slides. Post acclimation for 2-3 days, treat with TGFb2 (5 ng/mL) or vehicle (PBS) for 7-10 days, replacing with fresh medium twice per week.
2.3 Visualization and quantification of CLANs in confluent primary TM cell cultures (Figures 2A-B and 3A, C-D)
- Human primary NTM or GTM cells are cultured as described above
- In confluent NTM or GTM cells or after DEX or TGFb2 treatment, fix cells with 2-4% paraformaldehyde in PBS and wash three times with PBS (Note 2).
- Permeabilize cells by treating with 0.1 - 0.5 % Triton X-100 in PBS for 10 mins then wash three times with PBS.
- Perform blocking for two hours at room temperature by using Superblock blocking buffer (ThermoFisher) or alternatively use 10% goat serum in PBS.
- Stain cytoplasmic F-actin using phalloidin conjugated with Alexa-488 (1:100; Invitrogen) diluted in Superblock blocking buffer for 1 hour at room temperature. If concurrent immunolabeling of other markers is required, stain overnight with primary antibody at 4 °C followed by washing and addition of the relevant fluorescently labelled secondary antibody.
- Wash three times with PBS and mount the glass coverslips using ProLong Gold Anti-Fade with 4′,6-diamidino-2-phenylindole (DAPI; Life Technologies) for nuclear counterstaining.
- Visualize CLANs using an epifluorescence or confocal microscope capable of achieving 600X total magnification. Refer to the representative images (Figure 2) to aid in identification of CLANs. CLANs are defined as F-actin–containing cytoskeletal structures with at least one triangulated actin arrangement consisting of actin spokes (struts) and at least three identifiable hubs (vertices) (Figure 2A). Linear fibers of F-actin bundles (i.e stress fibers) may be more prominent and prevalent in the images, masking the lighter stained CLANs which generally are above the stress fibers. As a result, higher magnification and manual focusing may be required to properly visualize CLANs from the highly fluorescent F-actin linear stress fibers. Capture and evaluate multiple images from each cover slip (take images at ~10 locations with equal cell populations) and 6-10 coverslips per treatment group (generally 100-200 TM cells/slide).
- Using the images taken, count CLANs in the images in a masked manner. In each image (field of view) count the number of DAPI-positive cells and CLAN-positive cells (CPCs). CLAN-positive cells (CPCs) are defined as any cell in the field of view containing at least one CLAN or multiple CLANs. The formation of CLANs can be compared using the percentage of CPCs, which is calculated by dividing the number of CPCs by the number of DAPI-positive cells.
2.4 Visualizing CLANs in situ and ex vivo in human ocular TM tissues
Visualizing CLANs in TM tissues is more challenging due to the complex 3D structure of the TM, where cells surround the ECM beams and cells within the JCT are somewhat randomly arranged. We have used donor eyes from control and POAG patients [20] as well as our ex vivo perfusion cultured human anterior segment model [24] to visualize CLANs in TM tissues (Figures 4A-C).
2.4.1 Visualizing CLANs in human donor eyes in situ [20] (Figure 4A)
- Hemisect the eye along the equator about 5 mm posterior to the limbus, separating the anterior from the posterior segment. Remove and discard the lens, iris, and the posterior portion of the ciliary body from the anterior segment using a pair of fine dissecting scissors. In a 6-well plate, immersion fix the remaining anterior segment for 24 hrs using 4% neutral-buffered formaldehyde, ensuring the entire tissue is submerged in the fixative [20].
- After fixation, use a surgical stab blade to make slicing cuts posterior and anterior to the TM towards the Schlemm’s canal. These slices are to be made across the entire length of the TM. Using a fine-pointed forceps, lift and peel the sliced tissue strips containing the TM and place each segment into a well of a 24-well plate.
- Wash the strips three times with 500 mL of PBS for 10 mins each. In the same 24-well plate, incubate the strips overnight at 4 °C in the dark with phalloidin-Alexa 488 diluted 1/20 with PBS/0.1% Tween 20. After overnight incubation, again wash the strips three times with 500 mL of PBS for 10 mins each [20]. Incubate samples for 4 hours in propidium iodide (diluted 1/10 with 0.1% PBS) and wash an additional 3 times with 0.1% PBS.
- Carefully transfer the TM strip to a glass microscope slide to be mounted beneath a coverslip in fluorescence mounting medium. Ensure the orientation of the strip is similar along the entire length of the TM strip.
- Use a confocal microscope with a 60X oil immersion objective (NA = 1.4) for visualizing CLANs in ex vivo tissues. Optically section the strips at 0.5 – 1 µm intervals into a z-series of confocal images. Note that the phalloidin-Alexa 488 will fluoresce comparatively more when bound to larger linear actin stress fibers compared to the subcortical CLANs. Manual focus adjustment may be necessary to augment the visibility of CLANs from the linear cortical actin stress fibers during imaging.
- Use confocal microscope software for processing and evaluating the individual images of the z-stack to identify and determine the size, complexity, and prevalence of CLANs in each field of view. For all the CLANs that occupy more than one z-series (1 µm) image, align the stack images in register; and create composites for analysis. This provides a more robust inclusion/exclusion strategy for CLAN identification and a more precise measurement of the dimensions of these structures (Figure 4A). Also be mindful of intraobserver and interobserver variability that can occur in counting CLANs.
- For quantification, calculate the number of CLANs and number of PI-stained nuclei in a known volume of TM tissue. TM volumes were determined from the area of confocal z-stacks times the number and depth of each optical slice. Be aware that there is an age-related decline in TM cell number [43, 44] that is further acerbated in POAG eyes [45], so that it is important to age-match eyes.
2.4.2 Visualizing CLANs in the TM of ex vivo perfusion cultured anterior segments [24]
- Ex vivo human anterior segment perfusion culture: The technique of ex vivo perfusion culture of human anterior segments varies among labs, but we have been using slight modifications from the original technique [39, 46-50] initially described by Doug Johnson [51]. Anterior segments are dissected from human donor eyes (<24 hours post death), with careful removal of the lens, iris, and the majority of the ciliary body. Each anterior segment of paired eyes is placed on an island and clamped with an O-ring in a specially engineered Plexiglass dish. One dish cannula is attached to a syringe pump and the other canula to pressure transducer to continuously monitor IOP. The covered perfusion dishes are placed in an incubator at 37oC with 5% CO2/air. Perfusion medium (low glucose DMEM containing L-glutamine and pen/strep) is perfused at the constant rate of 2.5 mL/min (approximately the rate of aqueous formation). The IOPs generally stabilize after 24-48 hours of perfusion culture. To induce CLANs, one eye of each pair receives either 100 nM DEX (in 0.1% ethanol) or TGFb2 (5 ng/mL), while the contralateral eye receives vehicle. Perfusion continues for 7-14 days. Approximately 30-40% of the DEX perfused eyes develop significantly elevated IOP (at least 5 mmHg elevation above baseline) [46], while almost all eyes receiving TGFb2 develop OHT [39]. Alternatively, TM tissues can be transduced with either Ad5 or lentiviral vectors encoding human TGFb2C226/228C to generate TGFb2-induced ocular hypertension.
- Visualization of CLANs [24] (Figures 4B-C): CLANs within the TM, JCT, and inner wall of Schlemm’s canal (SC) can be assessed using two methods. To visualize the uveal and corneoscleral TM, human anterior segments are perfusion fixed at 12 mmHg for 1 hour and then immersion fixed for 23 hours in neural buffered 4% formalin. The TM tissue is then processed as described above (2.4.2.1). The second method examines the JCT and SC inner wall (IW) endothelium. Anterior segments are perfusion fixed for 2 hours and then immersion fixed for 22 hours in PBS/3% paraformaldehyde. Radial segments for each quadrant are microdissected, making an incision along the posterior margin of SC and opening SC to allow en-face visualization of the inner wall endothelium and adjacent JCT. Tissues are permeabilized in 0.2% Triton X-100 in Dulbecco’s PBS for 5 minutes and subsequently washed in DPBS. Tissues then are stained with rhodamine-phalloidin (diluted 1:20 in DPBS) for 30 at 37oC, and nuclei are stained with Sytox Green (1:2,400 in TBS) for 5 minutes at room temperature. After washing issues in DPBS, a thin piece of tissue containing SC IW, JCT and remaining meshwork is dissected from remnants of ciliary body and iris root. The thin tissue samples are mounted on a slide containing a shallow chamber constructed from Parafilm to prevent tissue compression when adding the cover slip. Mounted tissues are examined by confocal microscopy using the 60X oil immersion lens and optically sectioned at 0.5-1.0um intervals to generate Z-stacked images from SC IW through the JCT and into the corneoscleral TM.