A method describing the microdissection of trabecular meshwork tissue from Brown Norway rat eyes

Abstract

Glaucoma is often associated with elevated intraocular pressure (IOP), generally due to obstruction of aqueous humor outflow within the trabecular meshwork (TM). Despite many decades of research, the molecular cause of this obstruction remains elusive. To study IOP regulation, several in vitro models, such as perfusion of anterior segments or mechanical stretching of TM cells, have identified several IOP-responsive genes and proteins. While these studies have proved informative, they do not fully recapitulate the in vivo environment where IOP is subject to additional factors, such as circadian rhythms. Thus, rodent animal models are now commonly used to study IOP-responsive genes in vivo. Several single-cell RNAseq studies have been performed where angle tissue, containing cornea, iris, ciliary body tissue in addition to TM, is dissected. However, it is advantageous to physically separate TM from other tissues because the ratio of TM cells is relatively low compared to the other cell types. In this report, we describe a new technique for rat TM microdissection. Evaluating tissue post-dissection by histology and immunostaining clearly shows successful removal of the TM. In addition, TaqMan PCR primers targeting biomarkers of trabecular meshwork (Myoc, Mgp, Chi3l1) or ciliary body (Myh11, Des) genes showed little contamination of TM tissue by the ciliary body. Finally, pitfalls encountered during TM microdissection are discussed to enable others to successfully perform this microsurgical technique in the rat eye.

Keywords: Microdissection; RNA isolation; Trabecular meshwork.

Figure 1.. Histology of tissues comprising the rat conventional outflow pathway.

(A, B) Methylene blue and fuschin staining of a rat anterior segment embedded in epoxy. S=sclera, CC=collector channel, CB=ciliary body, C=cornea, AC=anterior chamber, SC= Schlemm’s canal, and TM=trabecular meshwork. Image modified from (Morrison et al., 2011).

Figure 2.. Microdissection of rat trabecular meshwork tissue.

(A) The anterior segment was removed and cut into quarters (B). (C) The iris and ciliary body were removed with fine jeweler’s forceps to expose the TM. Arrows indicate dissection pins used to anchor the cornea and sclera to the underlying wax. (D) Speckled TM (*) lies between pigmented Schwalbe’s line (white arrows) and ciliary body remnant (white arrowheads) attached to the sclera. (E) The TM (white arrow) was gently peeled off, revealing the white outer wall of Schlemm’s canal (*). (F) Region without TM (white bracket) contrasts with region still containing TM (black bracket).

Figure 3.. Movie showing how the jeweler’s forceps are sharpened.

Figure 4.. Movie showing the microdissection of rat trabecular meshwork tissue.

Figure 5.. Histology and immunohistochemistry of rat anterior segments.

(A, C) Anterior segment tissue pre-TM dissection. (B, D) Anterior segment tissue after TM microdissection. Representative images from (A, B) H&E stained paraffin sections and (C, D) α-smooth muscle actin immunostained (green) sections. DAPI stained the nucleus (blue). Note the minimal staining in D where the TM attaches to the sclera (arrow), indicating that the majority of the TM tissue was removed. TM= trabecular meshwork; SC=Schlemm’s canal; SL=Schwalbe’s line. Scale bar = 20 μm.

Figure 6.. TaqMan PCR to quantitate TM and CBI biomarkers in each tissue.

(A) Mgp, Myoc, and Chi3l1 are TM biomarkers, while (B) Des and Myh11 are CBI biomarkers. Results are from n=6 eyes from n=5 biological replicates. Mgp, p=0.033; Myoc, p=0.0001; Chi3l1 and Des, not significant; Myh11, p=0.0025.