Download Title: “Ultrastructure of the anterior lens capsule`s epithelium in

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 in lens capsule’s epithelium
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.
Image 1. Photos TEM A. x6000, B.x15000. Part of the basement membrane (up left) and 2 epithelial cells
are detected. Diffuse intracellular edema is observed, more intense in the cell below. Localized intracellular
edema appearing as empty spaces. The nuclei of the cells have microgranular structure.
Image 2. Photo TEM A. x5000, B. x10000. Part of the basement membrane (below). Cell with diffuse
intracellular and localized extracellular edema. Apoptotic nucleus. Cytoplasmatic projection of another cell
above the free surface of the cell with the apoptotic nucleus.
Image 3. Photo TEM x 10000. Part of the basement membrane (below) and cells with a varying degree of
diffuse and localized intracellular edema. Intercellular edema. Loss of cuboidal shape of the cells.
Cytoplasmatic projections of other cells over the free surface of the epithelium, multilayering.
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