Simple fixation and storage protocol for preserving the internal structure of intact human donor lenses and extracted human nuclear cataract specimens

摘要

Purpose: Increased use of phacoemulsification procedures for cataract surgeries has resulted in a dramatic decrease in the availability of cataractous nuclear specimens for basic research into the mechanism of human cataract formation. To overcome such difficulties, a fixation protocol was developed to provide good initial fixation of human donor lenses and extracted nuclei, when available, and is suitable for storing or shipping cataracts to laboratories where structural studies could be completed. Methods: Cataractous lens nuclei (n=19, ages 12 to 74 years) were obtained from operating suites after extracapsular extraction. Transparent human donor lenses (n=27, ages 22 to 92 years) were obtained from the Ramayamma International Eye Bank. After the dimensions were measured with a digital caliper, samples were preserved in 10% formalin (neutral buffered) for 24 h and followed by fixation in 4% paraformaldehyde (pH 7.2) for 48 h. Samples were stored cold (4 °C) in buffer until shipped. Samples were photographed and measured before further processing for transmission electron microscopy. Results: The dimensions of the samples varied slightly after short fixation followed by 1 to 5 months’ storage before transmission electron microscopy processing. The mean change in the axial thickness of the donor lenses was 0.15±0.21 mm or 3.0±5.4%, while that of the extracted nuclei was 0.05±0.24 mm or 1.8±7.6%. Because the initial concern was whether the nuclear core was preserved, thin sections were examined from the embryonic and fetal nuclear regions. All cellular structures were preserved, including the cytoplasm, complex edge processes, membranes, and junctions. The preservation quality was excellent and nearly equivalent to preservation of fresh lenses even for the lens cortex. Cell damage characteristic of specific nuclear cataract types was easily recognized. Conclusions: The novel fixation protocol appears effective in preserving whole donor lenses and cataractous nuclei over a wide age range. Dimensions varied only 2%–3%, and fiber cell damage correlated well with standard fixation. These methods enable researchers and clinicians in remote settings to preserve donor lenses and rare examples of extracapsular extractions for detailed examination at later times.