The effect of paraformaldehyde fixation and PBS storage on the water content of the human lens

Purpose: Fixation and phosphate buffered saline (PBS) storage are frequently used before studies of the morphological, biochemical, and optical properties of the human lens begin. It is assumed that this does not alter the properties being examined. The present study was undertaken to determine the effects of fixation and PBS storage on the human lens wet weight. Methods: Human donor lenses were incubated in a buffered paraformaldehyde (PF) solution or in PBS and their wet weights were monitored for up to 44 and 13 days, respectively. Results: PF fixation resulted in a large decrease in wet weight, averaging 25%±2.3% at 30 days for 14 human donor lenses, aged 49–80 years. The loss was essentially complete by 21 days. Out of the 10 lenses, aged 52–71 years, which were incubated in PBS alone, six of them increased in weight by an average of 38% over 13 days and four ruptured within four days. Comparison of literature data for a fixed eight-year-old lens with those for an unfixed seven-year-old lens indicated that the decrease in wet weight was due mainly to a loss of water from the cortex, which resulted in virtual disappearance of the water/protein gradient and the formation of a plateau containing 58% water in over 90% of the lens. Conclusions: Fixation substantially alters the amount and distribution of water in the human lens. Caution should be exercised when interpreting data on water and protein distributions as well as cell dimensions obtained with lenses which have been fixed. In addition, prolonged storage of a lens in PBS will result in substantial water uptake, which may affect measurements of their dimensions and optical properties. The ocular lens grows continuously throughout life by mitotic division of the epithelial cells in the anterior preequatorial region. The post-mitotic cells subsequently differentiate and elongate to form new cohorts of fiber cells, which cover their predecessors. Unlike in other tissues of ectodermal origin, the ‘older’ cells are not shed and retain the majority of their proteins (crystallins). Instead, they are progressively compacted and form the different parts of the lens nucleus. This compaction is thought to be due to the loss of water. As a consequence, the concentration of protein and, hence, the refractive index gradually increases. This generates a refractive index gradient, increasing from the outside to inside, which is essential for reducing spherical aberration of the lens. One of the key requirements for understanding the biologic origin of lens optical properties, such as the refractive index, and how these properties change during the development of cataracts and presbyopia is a detailed knowledge of the content and distribution of water and protein in the lens. Several attempts have been made to obtain such