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M J. Costello, Ashik Mohamed, Kurt O. Gilliland, Sangeetha Metlapally, Sönke Johnsen, W C. Fowler; Simple Fixation and Storage Protocol for Preserving the Internal Structure of Intact Donor Lenses and Extracted Human Nuclear Cataracts. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3031.
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© ARVO (1962-2015); The Authors (2016-present)
Increased use of phacoemulsification for cataract surgery has resulted in a dramatic reduction in the availability of cataractous nuclei for basic research into the mechanism of human cataract formation. To overcome some of these difficulties, a fixation protocol was developed to give good initial fixation of donor lenses and extracted nuclei, when available, that is suitable for storage or shipment to laboratories where structural studies could be completed.
Cataractous lens nuclei were obtained from the operating suites at LVPEI after extracapsular extraction (n = 19, ages 12-74). Transparent donor lenses were obtained from the Ramayamma International Eye Bank, LVPEI (n = 27, ages 22-92). After dimensions were measured using a digital caliper, samples were preserved in 10% formalin (neutral buffered) for 24 hrs, followed by fixation in 4% paraformaldehyde (pH 7.2) for 48 hrs. Samples were stored cold (4°C) in buffer until shipped. Samples were photographed and measured prior to further processing for transmission electron microscopy (TEM) using methods described previously (Costello et al, EER 2010).
Sample dimensions varied slightly after short fixation followed by 1-5 mo storage before TEM processing. Average axial thickness change of donor lens = 0.15 ± 0.21 mm or 3.0 ± 5.4% (mean ± SD; n = 27); extracted nuclei average thickness change = 0.05 ± 0.24 mm or 1.8 ± 7.6% (mean ± SD; n = 19). 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 quality of preservation was excellent and nearly equivalent to preservation of fresh lenses. Cell damage characteristic of specific cataract types could be easily recognized.
The novel fixation protocol appears to be 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.
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