Using DNA and ROS specific fluorescent dyes and dual laser confocal microscopy,
11 we have shown that the development of subcapsular and cortical cataract in old mice of an inbred strain (C57BL/6) and also an outbred strain of mixed genealogy correlates with the retention of undigested cellular debris and DNA in incomplete differentiating lens fiber cells. These accumulations arise from two sources: at the bow region, as a build-up of debris in lens fiber cells that fail to differentiate normally, and at other sites on the anterior epithelium, where strands of surface epithelium invade the subcapsular space and cortex. This light-refractive material consists of nuclear fragments, mitochondria, and free DNA. The DNA inclusions also contained ROS-positive material that was concentrated in mitochondrial remnants. All these items correlated spatially with each other and with light reflected from cataractous opacities. The strands of surface epithelium invading the anterior cortex were commonly derived from the central zone of the lens, a region that is normally without cell replication. Because the strands are attached to denuded lens surface, this invasion appears to represent migration of LECs from the surface down into the underlying subcapsular and cortical area, rather than aberrant replication, which would not by itself leave gaps on the surface.
Our findings can also be related to several recent reports in which retention of nuclear remnants was found in young rodents with specific cataractogenic mutations or after experimental cataractogenic treatments. Nishimoto et al.
27 have reported DNA accumulations, along with cataract formation, in the lenses of young mutant mice lacking the DNase II-related enzyme DLAD. These appear to be very similar to those we report in the nonmutant old cataract-bearing animals. A major difference is that the DLAD-deficient mice accumulated DNA early in their development, including within the lens nucleus, whereas, our studies demonstrate the accumulation late in life and only in the cortex and subcapsular regions. Much of the lens nucleus of the old mouse would have formed early in the mouse’s life before these age-related involutions and inclusions occurred. Therefore, age-related nuclear cataract, which is common in old mouse lenses, may not be related to the process we describe, although the age-related changes that we observed in the lens cortical areas might alter the internal milieu of the lens nucleus in a way that is conducive to nuclear cataract. In another report, Hegde and Varma
30 found that the streptozotocin-diabetic mouse appears to have an involution of surface anterior epithelium near sites of cataract development, and an unresolved bow region similar to that which we observed. Their findings include retention of nuclear material in incompletely differentiated lens fiber cells, especially in the anterior region of the lens, but also present in the posterior region. It is possible that the development of diabetic and age-related cataracts has some similarity in the physical events that produce both, whether or not the initiating process is similar. Finally, Vrensen et al.
31 report that cataract induced in young rats by tryptophan deficiency is associated with an extension of the bow region very similar to our observation. The invasion of the subcapsular and cortical region of the lens that we describe herein has some characteristics of the morphology produced by experimentally altered TGF-β
32 or Pax6 gene expression.
32 33 34 However, our studies differ with theirs in that we did not see conversion of epithelial cells to fibroblast-like cells, and they did not report an expanded bow region with retained nuclei and DNA. Definitive determination of whether these or other gene expression-driven changes occur in the aged mouse lens awaits further study. We are aware of the alterations in lens crystallins that occur with aging in the mouse (and other species).
22 We suggest that the age-related increase in ROS and the failure to complete cellular differentiation that we describe herein may contribute to such alterations in crystallins and lens fiber formation.
Another possibility regarding the failure of old lenses to degrade organelles is that the overall environment inside the old mouse lens is compromised and no longer sends the appropriate signals to LECs at the bow region and elsewhere, to maintain an orderly progression and to trigger terminal differentiation and degradation of their organelles. The interior of the mature lens has a very special environment that is low in oxygen and high in acid and that may be necessary for proper differentiation of lens fiber cells.
35 Loss of these special conditions inside the lens cortex may be conducive to the involution of the surface epithelial cells into the underlying cortex and delay in fiber cell differentiation that we observe. In support of the latter concept, our preliminary experiments using a pH-sensitive fluorescent probe (Lysotracker Red; Cambrex Corp., E. Rutherford, NJ) have indicated that the interior of the old lens is less acid than that of young control lenses, especially in the areas of DNA inclusions (our unpublished observations, 2005). Several of the mentioned possibilities and their relationship to the retention of nuclear and mitochondrial fragments are currently under further investigation. Also to be determined is whether the condition that we report in mice is present in other mammals, including humans. Indeed, in preliminary studies now under way, we are finding very similar changes in the lenses of aged pigmented rats (manuscript in preparation). A search of the literature on age-related cataract in the human did not yield reports with results that were in concordance with ours, specifically, epithelial gaps, LEC invasion, nuclear fragment retention, free DNA, and ROS presence in the region of cataract development. However, earlier studies using light, scanning, electron, and specular microscopy
23 reported granular material in the posterior subcapsular region of cataractous human lenses that may represent the nuclear debris that we report herein, and several studies report reduced numbers and outright loss of surface epithelial cells associated with subcapsular and nuclear cataractous lenses similar to our findings.
24 36 37 38 Because our findings in the rodent model may have relevance to human age-related cataract, we are beginning similar studies of monkey and human whole lenses.