A comparison of different embedding media on the ultrastructure of the trabecular meshwork.
OBJECTIVE
To determine whether the preservation of the extracellular matrix and the ultrastructural appearance of the trabecular meshwork are affected by different histologic processing protocols and embedding media. Conventionally used epoxy resins such as Araldite require complete dehydration of tissue, while the acrylic resin LR White requires only partial dehydration, better preserves tissue antigens, and has been reported to preserve more of the extracellular matrix.


METHODS
Seven human eyes ranging in age from 2 months to 78 years were dissected and tissue samples from each eye processed and embedded in both Araldite and LR White media. The ultrastructure of the trabecular cells and the extracellular matrix of the meshwork was compared between media. The preservation of the extracellular matrix in the juxtacanalicular region was determined by measuring the amount of material immediately underlying Schlemm(1)s canal. Immunoelectron microscopy was used to determine the composition of this material.


RESULTS
Araldite provided better resolution of ultrastructural details than freshly polymerized LR White. After a period of ripening for several months, however, resolution of tissue details in LR White improved. No significant quantitative difference was found in the amount of extracellular matrix underlying Schlemm's canal when comparing the two media. Neither post-mortem time to fixation (up to 31 hr), donor age, nor immersion vs. perfusion fixation technique affected the amount of extracellular material present in the comparison of the two embedding media. Immunogold labeling of the extracellular material within the juxtacanalicular tissue revealed the presence of collagen IV, laminin, and fibronectin in the basement membrane region immediately underlying the inner wall, and also in scattered patches within the juxtacanalicular tissue.


CONCLUSIONS
Despite the less rigorous processing required for LR White than epoxy embedding, neither the appearance nor amount of the extracellular material was affected by the different embedding protocols. Prolonged post-mortem time to fixation did not affect the appearance nor amount of extracellular matrix. Immunolabeling revealed that the extracellular matrix of the juxtacanalicular tissue contains components of basement membrane material.
Brilakis
H S
HS
Department of Ophthalmology, Mayo Clinic and Mayo Foundation, Rochester, MN 55905, USA.
Hann
C R
CR
Johnson
D H
DH
D000180:Acrylic Resins; D000368:Aged; D004853:Epoxy Resins; D005109:Extracellular Matrix / Q000648:ultrastructure; D006801:Humans; D007223:Infant; D008875:Middle Aged; D010796:Phthalic Anhydrides; D016610:Tissue Embedding / Q000379:methods*; D014021:Tissue Preservation; D014129:Trabecular Meshwork / Q000648:ultrastructure*
D000180:Acrylic Resins; D004853:Epoxy Resins; D010796:Phthalic Anhydrides; C005752:araldite; C048707:LR white