Contractile markers distinguish structures of the mouse aqueous drainage tract

Animals

Animal care and use was in compliance with institutional guidelines and with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. C57BL/6 and BALB/c mice (n=6–8 mice of each strain, 6–8 weeks old) were purchased from Charles River Laboratories International, Inc. (Wilmington, MA). Animals were stored in a temperature-controlled room with a 12h:12h light-dark cycle and fed ad libitum.

Reagents

Alexa Fluor 568–conjugated phalloidin and ProLong Gold Anti-fade with 4',6-diamidino-2-phenylindole were purchased from Life Technologies (Grand Island, NY). Tissue-Tek optimum cutting temperature compound was purchased from Sakura Finetek (Torrance, CA). Paraformaldehyde, Triton X-100, and bovine serum albumin were purchased from Sigma (St. Louis, MO).

Antibodies

Alexa Fluor 488-, 568-, or 633–conjugated secondary antibodies were purchased from Life Technologies. Rabbit or goat anti-α-SMA (Cat # ab5694 or ab21027, 1:100 dilution), mouse anti-MHC nonmuscle (Clone 3H2, Cat # ab684, 1:200 dilution), rabbit anti-caldesmon (E89, Cat # ab32330, 1:200 dilution), and rabbit anti-calponin (Clone EP798Y, Cat# ab5694, 1:100 dilution) were purchased from Abcam (Cambridge, MA). Normal goat, rabbit, and mouse immunoglobulin G isotypes were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Immunofluorescence staining

Enucleated eyes were quickly embedded in Tissue-Tek optimum cutting temperature compound. Cryosections (7 µm thickness) were fixed with 4% paraformaldehyde and further permeabilized/blocked in the blocking solution (5% bovine serum albumin and 0.3% Triton X-100) for 1 h at room temperature. Sections were incubated overnight at 4 °C with combinations of primary antibodies (single, dual, or triple) in the blocking solution. The sections were further incubated for 1 h at room temperature with a combination of secondary antibodies, Alexa Fluor–conjugated anti-rabbit, mouse, and/or goat antibodies, and then mounted using ProLong Gold Anti-fade reagent with 4',6-diamidino-2-phenylindole. To visualize polymerized actin, sections were subsequently incubated with Alexa 568–phalloidin along with the appropriate combination of secondary antibodies. Negative, nonspecific labeling was established with normal immunoglobulin G isotypes. Antibody specificity was confirmed by immunoreactivity in blood vessels and extraocular muscle in the same sections used for primary labeling, which provided internal positive controls. Sections were analyzed with a Leica SP5 high-speed spectral confocal laser-scanning microscope (Leica Microsystems, Wetzlar, Germany) or a Zeiss LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). Immunofluorescence staining for single or double contractile markers was performed using randomly selected slides (4–5 slides per each eye) containing four sections per slide and examined under the confocal microscope. Additional cryosections and formalin-fixed paraffin-embedded tissue sections (FFPE) were stained with hematoxylin and eosin (H&E) and examined by light microscopy for morphology.

Quantification and stereological counting procedure

Specific fluorescence from tissue labeling in histological sections was captured by confocal microscopy with exposure time kept constant across all images. Image sections were imported as 16 bit images and analyzed by NIH Image J software. Fluorescence intensity of pixel gray values in eight separate regions of interest (ROIs) per region of TM and CM was calculated and averaged across each tissue region. This was performed separately for each label (filamentous actin [F-actin], α-SMA, MHC, caldesmon, and calponin) in TM and CM and averaged across data from three mice. Fluorescence intensity for F-actin, α-SMA, MHC, caldesmon, and calponin in the TM and CM were then compared using analysis of variance (ANOVA) and Tukey’s and Sidak’s comparison tests.

Aqueous drainage tract histology in C57BL/6 mice

The TM begins anteriorly at the termination of the corneal endothelium, where the corneal endothelial monolayer transitions to a multilayered cellular structure in the iridocorneal angle, as shown in Figure 1. This part of the TM becomes pigmented near the apex of the iridocorneal angle and iris root. SC starts just posterior to the iridocorneal angle and adjacent to the pigmented TM in the angle. The TM continues as a multilayered cellular structure right next to SC deeper in the inner sclera until SC terminates posteriorly just beyond the ciliary processes.

The CM is identifiable posteriorly near the anterior termination of the retina. It borders ciliary body (CB) pigment. The expected anterior extension of the CM in between the tissue planes of the pigmented CB and TM is hard to identify in H&E sections.

Filamentous actin labeling in C57BL/6 mice

In C57BL/6 mice, phalloidin labeling of F-actin in the aqueous drainage tract was strongly positive in a band, consistent with CM adjacent to the pigmented CB, as shown in Figure 2A. CM F-actin labeling was broadest and most intense posteriorly near the anterior termination of retina. More anterior CM labeling extended alongside the pigmented CB (open arrows). TM F-actin labeling was seen in the TM region adjacent to the corneal endothelium and in inner scleral aspects of the TM adjacent to SC (closed arrows).

Overall, F-actin labeling was seen in both CM and TM, albeit less intensely in TM. It was hard to distinguish between the structures of TM and CM by F-actin labeling alone.

α-Smooth muscle actin and caldesmon immunolocalization in the ciliary muscle and trabecular meshwork

Positive α-SMA labeling was seen in the CM, as shown in Figure 3A. α-SMA labeling formed a bright band in the posterior regions of the CM (open arrows) and a thin and wavy structure adjacent to the pigmented CB and anteriorly near the iridocorneal angle (arrowhead; Figure 3A). α-SMA labeling was positive but spotty in the cellular layers of the TM next to SC. F-actin and α-SMA labeling clearly colocalized in the CM. F-actin and α-SMA co-localization was patchy and less apparent in TM cellular layers adjacent to SC (dotted outline in Figure 3F; high power).

Positive caldesmon labeling was seen in the TM (closed arrows; Figure 4A) and CM (open arrows; Figure 4A). In the CM, caldesmon labeling was present in its posterior portion and more anteriorly next to the pigmented CB, seen as a thin streak extending to the iridocorneal angle. Weaker, speckled caldesmon labeling was present in TM cellular layers next to SC, and also more anterior portions of TM starting at the terminus of corneal endothelium. F-actin and caldesmon colocalization was seen primarily in the CM (yellow; Figure 4C,F), with colocalization spotty in TM cellular layers adjacent to SC (dotted outline in Figure 4F; high power).

Non-muscle myosin heavy chain immunolocalization in the trabecular meshwork

Positive non-muscle MHC labeling was primarily observed in the TM (closed arrows; Figure 5A), as seen in cellular layers adjacent to SC and the corneal endothelium terminus at the iridocorneal angle (arrowhead; Figure 5A). MHC labeling intensity was relatively low in the CM, especially posteriorly, relative to positive F-actin labeling in the CM (opened arrows; Figure 5C,F). Patchy co-localization of MHC and F-actin was seen in TM cellular layers adjacent to SC (dotted outline; Figure 5C,F).

Calponin immunolocalization in ciliary muscle

Positive calponin labeling was seen in the CM (open arrows) posteriorly and in more anterior CM regions adjacent to the pigmented CB, as shown in Figure 6. Calponin colocalized with F-actin in the CM. Very limited or no calponin labeling was present in TM (closed arrows) cellular layers next to SC, where only occasional spotty calponin colocalization with F-actin was seen (next to the dotted outline of SC; Figure 6E,F)

Primary immunolocalization of α-smooth muscle actin, caldesmon, nonmuscle myosin heavy chain, and calponin distinguish ciliary muscle and trabecular meshwork

Primary immunolocalization of α-SMA (Figure 7A,E), caldesmon (Figure 7B), calponin (Figure 7I), and nonmuscle MHC (Figure 7F,J) were exhibited in different regions of the aqueous drainage tract. α-SMA labeled the CM primarily, extending from a broad band of CM labeling posteriorly to a thinner, wavy band of labeling anteriorly and adjacent to the pigmented CB, as shown in Figure 7A,E. α-SMA and caldesmon colocalization was seen primarily in the CM, with spotty colocalization in the TM (Figure 7C). α-SMA and calponin labeling in TM cellular layers adjacent to SC were of low intensity and speckled, and colocalization with MHC here was minimal and spotty (Figure 7G,K). Hence, primary immunolocalization of α-SMA and calponin (primarily the CM), and of MHC (primarily the TM) was mostly separate, allowing structures of the CM and TM to be distinguished in the aqueous drainage tract. Immunolocalization of these markers in the CM (opened arrows) was distinct from that of nonmuscle MHC, which primarily labeled TM (closed arrows), as shown in Figure 7K.

Contractile markers in trabecular meshwork and ciliary muscle of albino BALB/c mice

Contractile marker labeling that was performed in the C57BL/6 aqueous drainage tract was repeated in albino BALB/c mice lacking pigment in order to determine whether pigment obscured interpretation of the CM. In BALB/c mice (n=6), a reticular pattern of F-actin and MHC was seen in the CB, which was otherwise obscured by pigment in C57BL/6 mice (Figure 8A,B,F,J,N). The pattern of α-SMA, caldesmon, and calponin labeling in the CM of BALB/c mice was similar to that in C57BL/6 mice, confirming that pigment did not obscure interpretation of the CM in C57BL/6 mice (Figure 8E,I,M). Colocalization of F-actin with α-SMA, caldesmon, and calponin was mainly seen in the CM (open arrows), while MHC showed partial colocalization with F-actin in the CM and TM (Figure 8C,D,G,H,K,L,O,P), confirming that the colocalization pattern of contractile markers was similar in both C57BL/6 and BALB/c mice.

Negative controls for each antibody were isotype controls and controls in which the primary antibody was omitted; these showed no specific reactivity (data not shown). Antibody specificity was confirmed in internal positive controls in the same sections. This comprised positive labeling of the intramural smooth muscle of extraocular blood vessels, wherein specific immunoreactivity was confirmed (data not shown).

Features of contractile markers in the trabecular meshwork and ciliary muscle

Figure 9 shows the extent of positive labeling for contractile markers, as quantified by fluorescence intensity measurements for each label in the aqueous drainage tissues of C57BL/6 mice. In the CM, mean fluorescence intensity was greatest for F-actin and least for MHC. CM MHC fluorescence intensity was significantly less than that of F-actin (p=0.2; one-way ANOVA and Tukey’s comparison test). In the TM, mean fluorescence intensity was greatest for MHC and least for calponin. The TM fluorescence intensities of α-SMA, caldesmon, and calponin were less than those of F-actin and MHC (p=0.0006; one-way ANOVA and Tukey’s comparison test). Fluorescence intensities of F-actin and classic smooth muscle markers, caldesmon, α-SMA and calponin, were significantly higher in the CM compared with the TM (p<0.0001; two-way ANOVA and Sidaki’s comparison test). MHC fluorescence intensity in the TM and CM was similar (p>0.05).