Title: “Ultrastructure of the anterior lens capsule’s epithelium in patients with age-related cataract” PURPOSE. The aim of this study is to locate, describe and evaluate the ultrastructural alterations of the anterior lens capsule’s epithelium in patients affected by cataract and to connect them with findings in previous studies.
METHODS. Capsulotomy specimens of four patients, over 60 years old, who have undergone phacoemulsification, were used after continuous curvilinear capsulorhexis. These were studied by means of Transmission Electron Microscope (TEM).
RESULTS. The examination of the lens epithelia revealed a diffuse intracellular edema, along with an extracellular edema in varying degrees. The cells were loosely connected amongst them and with the basement membrane. Very often there was more than one layer of cells, covering the damaged, underlying cells. It was a common finding that cells lost their regular shape and were often presented with expanded nuclei carrying dense granules and swollen vesicles. In all the samples apoptotic nuclei were detected.
CONCLUSION.The present study describes alterations of the lens capsule epithelial cells that may be characterized as degenerative and connects them with the existing information in the literature. They are possibly associated with the age-related cataract, since the patients have no other known medical history.
INTRODUCTION Human eye lens consists of three main parts: the lens capsule, the lens epithelial cells and the lens fibers. The lens epithelium is located exclusively on the anterior side of the lens and is normally a simple cuboidal epithelium. Those cells contribute to the homeostasis of the lens, incorporating a Na+/K+-ATPase which pumps ions, nutrients and liquid into the lens and lens fibers (Seland, 1974).
A frequent disease of the eye is cataract, which creates opacities of the lens leading to a decrease in vision. Considering the important functions attributed to the lens epithelium, its damage has been an area of interest for researchers attempting to discover a possible connection to cataract formation. Ageing is the most common cause of cataract, but the disease can also occur due to trauma or other conditions such as diabetes mellitus or radiation exposure or even as a congenital defect (Harocopos et al., 1998). This study concentrates on the characteristics of the lens epithelium that are probably connected to the age-related cataract. The purpose of this study is to identify possible unrecorded features and to compare all new observations with previous studies.
METHODS Five patients with age-related cataract, older than 60 years, were included in the study. The research was approved by the Ethical Committee of the Aristotle University of Thessaloniki. Anterior lens capsules (aLCs) were obtained from patients during uneventful phacoemulsification by the same surgeon at Papageorgiou University Hospital in Thessaloniki. The 5-5.5 mm circle of the central aLC was carefully removed by continuous curvilinear capsulorhexis with forceps and was immediately prepared for transmission electron microscopy, performed in the Laboratory of Histology and Embryology of Aristotle University of Thessaloniki.
All the specimens were immediately fixed in a solution of a neutral buffered 3% gloutaraldehyde for 90 minutes and postfixed in 2% OsO4. All capsules were then dehydrated in increasing concentrations of ethanol and embedded in Epon 812. Semi-thin sections (1-3 μm) were made, stained with 1% cyane toluidine and analysed by light microscopy. Ultrathin sections (30-50 μm) were stained with uranyl-acetate and lead-citrate and examined by a Joel JEM-1011 transmission electron microscope.
RESULTS Degenerative changes of the lens epithelium were observed in all specimens. The examination of lens epithelia revealed a diffuse intracellular edema, along with an extracellular edema in varying degrees. Transparent vacuoles were detected between the cells and between the cells and the basic membrane. The cells were loosely connected amongst them and with the basement membrane. Very often there was more than one layer of cells, covering the damaged, underlying cells, maybe in an attempt to compensate. The cytoplasmic process that covered the cells always exhibited a higher density than the underlying cells. It was a common finding that cells lost their regular shape and were often presented with expanded nuclei carrying dense granules and swollen vesicles. In all the samples apoptotic nuclei were detected. The cell membranes were often ruptured and lost their regularity. The free epithelial cell surface very often exhibited a highly irregular margin.
DISCUSSION Review of the literature, description of cataractic capsules in EM
In 1980, Jensen and Laursen studied the anterior lens structures in patients with senile cataract, with special reference to anterior capsular/subcapsular opacity (ACSCO). In TEM, numerous large intercellular and a few intracellular vacuoles were encountered in lens epithelium in both ACSCO and non ACSCO group. Nuclei were observed to be compressed into irregular shapes by vacuoles in both groups (Jensen & Laursen, 1980). Exo- or endocytotic vesicles were found adjacent to the capsular as well as the cortical side of the epithelial cells (Laursen & Fledelius, 1979). Nuclei appearing with irregular shapes due to the compression of epithelial vacuoles have also been described previously (Hess, 1905), (Vogt,1914).
Similarly to the above-described observations, in our study, intracellular and intercellular vacuoles, influencing the appearance of both the nucleus and the whole cell, were observed in all patients, none of which had ACSCO type of cataract.
In electron microscopy studies of anterior lens epithelium of patients with intumescent white cataract, LEC swelling and degeneration have been described. Moreover in those epithelia, spherical formations were observed on the apical side of LEC's, towards the lens cortex. They are described bulging out from the apical cell membrane of the LEC's and disrupting it (Hawlina et al., 2011), (Andjelic et al, 2015). Although none of our patients suffered from intumescent white cataract, the respective degeneration lesions observed with TEM were more severe. On the other hand, no extrusions or bulges were observed in our study.
It was also reported that in senile lenses there is an abnormal migration and multilayering of cells, and a wrinkling of the lens capsule, a finding similar to ours (Rahim & Iqbal, 2011). Although there is a similarity to our results, this observation was described on an optical microscope study, which is therefore less detailed and accurate than TEM. To our knowledge there is no such description on an electron microscope.
Moreover Bleckmann et al in a study of cataractous anterior lens capsule and epithelium, mentioned proliferative changes with a multilamellar arrangement of the LECs and an increase in the pleomorphic appearance of the cell and nucleus. Examination of the lens capsule revealed intracapsular foamy and vacuolic deposits with a localized splitting of the lens capsule. (Bleckmannet al, 1989). All these descriptions are also optical microscope findings and have not been described by TEM, although an electron microscope was also used in the same study.
A loss of cell membrane integrity was reported using a fluorescent stain for DNA, BOBO-3, in cataractic capsules (Harocopos et al, 1998).
Apoptosis, i.e. programmed cell death, is considered a vital component of normal cell turnover, embryonic development, proper functioning of the immune system and often occurs in response to chemical or toxic agents. In the course of eye development apoptosis is beneficial to the separation of the lens from the future corneal epithelium as well as to the formation of the retina. The integrity and normal function of the tissue requires apoptosis in certain levels. However uncontrolled cell death results in pathological conditions.
Li et al. used the TUNEL method and discovered TUNEL-positive cells in capsulotomy specimens from patients who had undergone cataract surgery, but not in other, non cataractic samples, from eye bank eyes (Li et al, 1995). They concluded that apoptosis in the lens epithelium may promote noncongenital cataract formation.
On the other hand, another study argues that the TUNEL –positive cells in capsulotomy specimens came as a result of necrosis rather than apoptosis. They concluded that the loss of lens epithelial cells, either by apoptosis or by other mechanisms of cell death, does not seem to play a major role in age-related cataract formation (Harocopos et al,1998).
The apoptotic nuclei detected in our samples verifies apoptosis does indeed exist in epithelial capsular cells. We cannot however establish a correlation between apoptosis and cataract.