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Immunohistochemical characteristics of the vitreolenticular interface in congenital unilateral posterior cataract

Van Looveren, Jan MD, FEBO*; Van Gerwen, Veerle MSc; Timmermans, Jean-Pierre PhD; Tassignon, Marie-José MD, PhD, FEBO

Author Information
Journal of Cataract & Refractive Surgery: July 2016 - Volume 42 - Issue 7 - p 1037-1045
doi: 10.1016/j.jcrs.2016.04.026
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Abstract

Although surgical treatment of cataract in children has improved substantially as a result of the rapidly increasing knowledge of pediatric cataract in general, little is known about the underlying pathophysiology of congenital unilateral cataract. These cataracts are most often of unknown underlying etiology (not hereditary, metabolic, or infectious) in otherwise healthy babies.1 Quite often, these cataracts do not display any of the typical signs of persistent fetal vasculature (PFV), such as a retrolenticular fibrovascular membrane or a persistent hyaloid stalk.2 Lens removal in these cataracts is often associated with a posterior capsule plaque, which is a dense white opacity that is strongly adherent to the internal surface of the posterior lens capsule.3 Based on clinical observations during cataract surgeries, Müller-Eidenböck et al.4 hypothesized that all infantile unilateral cataracts presenting with a posterior capsule plaque are caused by minimal fetal vascular remnants, a mild variant of PFV. They argued that the posterior lens capsule was invaded by fetal vessels, resulting in an abnormally strong attachment site between the hyaloid, posterior capsule, and cortex before dissipating. This hypothesis is supported by the results in the Infant Aphakia Treatment Study (IATS)5 video assessment of unilateral infantile cataracts, which showed an unexpectedly high incidence of posterior capsule plaques (88%) in unilateral congenital cataract and often increased cataract density in the posterior part of the fetal nucleus and cortex near the posterior capsule in association with posterior capsule plaque formation.

In normal eyes, the center of the posterior capsule and the anterior hyaloid membrane are separated by a virtual space called Berger space,6 which is normally fully formed by the ninth month of gestation. In PFV eyes, membrane-like structures between the posterior lens capsule and the retrolenticular fibrovascular membrane have been described in the literature.7,8 However, little is known about the vitreolenticular interface and its anomalies in pediatric cataract. Congenital posterior cataracts are believed to be triggered by abnormal development of the vitreolenticular interface, prompting us to further define the histological characteristics of this interface.4 Dysgenesis of the vitreolenticular interface might complicate posterior capsulorhexis, which is considered standard in pediatric cataract surgery.9,10 The integrity of the anterior hyaloid face cannot always be preserved, and sometimes even an unintended anterior vitrectomy is necessary.

Lens capsules contain high levels of collagen type IV (never collagen type II),11,12 whereas the vitreous chamber mainly contains collagen type II (but never collagen type IV).13 Thus, if collagen type II is present at the posterior surface of the posterior lens capsules, this provides evidence of dysgenesis of Berger space in this type of cataract.

Patients and methods

Sample Collection

Specimens of posterior lens capsules were collected during cataract surgery performed at Antwerp University Hospital, Edegem, Belgium. Bag-in-the-lens intraocular lens (IOL) implantation is a technique that requires a centered posterior capsulorhexis of 5.0 mm.14 After lens and cortex removal and before capsulorhexis initiation, the posterior lens capsule was separated from the anterior hyaloid membrane by injecting an ophthalmic viscosurgical device (sodium hyaluronate 1.0% [Healon]) into Berger space through a puncture hole in the posterior capsule (Figure 1, A). Posterior capsule plaques, if present, were left in place during this procedure. If this mechanical viscodissection was complicated because of strong vitreolenticular adhesions, a clinical diagnosis of dysgenesis of Berger space was made (Figure 1, B). After completion of the capsulorhexis, the specimens (5.0 mm in diameter) were removed from the eye, subsequently spread open on a lens glide, and immediately fixed in 4% paraformaldehyde for at least 2 hours at room temperature.

Figure 1
Figure 1:
A: Senile cataract with normal Berger space. Status after lens removal. Blue line defines the blister of the OVD injected into Berger space through a puncture hole in the posterior lens capsule. B: Congenital cataract with posterior capsule plaque and central vitreolenticular adherence. Status after lens removal. Blue line defines the blister of OVD injected through a puncture hole in the posterior lens capsule in an attempt to separate the posterior capsule and the anterior hyaloid.

Immunohistochemistry

After fixation, the posterior lens capsule specimens (including, if present, the posterior capsule plaque adhering to the inner surface of the capsule and the adhesions to the vitreous at the outer surface of the capsule) were dehydrated and embedded in paraffin. Paraffin sections that were 5 μm thick were soaked in xylene to remove the paraffin, rinsed in acetone, and hydrated in isopropanol (99%, 90%, 80%) and finally in distilled water.

After rehydration, samples used for collagen IV staining underwent antigen retrieval. Formalin-fixed tissue was briefly immersed in citrate buffer under pressure (10 mM sodium citrate [Sigma I4-2S1801] 0.05% Tween 20, pH 6.0) for a few minutes and then cooled for 15 minutes in phosphate-buffered saline (PBS) at room temperature. Sections for collagen II and collagen IV staining were permeabilized, and nonspecific protein binding was blocked with 0.1% Triton-X 100 in PBS 0.01 M pH 7.4, thimerosal 0.05%, sodium azide (NaN3) 0.01%, bovine serum albumin 0.1%, normal horse serum 10.0%, Triton X-100 1.0% (PAV/NHS) for 30 minutes at room temperature.

All sections were incubated overnight in a damp box at room temperature with primary antibodies against collagen type II (Abcam ab34712, dilution 1/50) and collagen type IV (Abcam ab6586, dilution 1/500), in accordance with the manufacturers’ instructions. These antibodies were diluted in PAV/NHS without 0.1% Triton X-100 (PBS 0.01M, thimerosal 0.05%, NaN3 0.01%, bovine serum albumin 0.1%, normal horse serum 10.0%). After 3 washes in PBS, secondary antibodies were applied for 2 hours at room temperature Again, 3 washing steps in PBS were performed to remove the unbound secondary antibodies. This was followed by the addition of 4′ 6-diamidino-2-phenylindole dihydrochloride (DAPI), 5 μg/mL in a PBS 0.01M pH 7.4 solution known to bind with double-stranded DNA (nucleus staining). After a final wash step, slides were mounted in mounting solution (Citifluor) to prevent photobleaching of fluorochromes. All samples were observed and photographed using a laser scanning confocal microscopy (LSM510 2-photon microscope equipped with 63× Plan-Apochromat, Carl Zeiss Meditec AG) with an argon laser line (488 nm) and a helium–neon laser line (543 nm) for visualizing the fluorescein isothiocyanate and indocarbocyanine labels. The DAPI was visualized using a titanium–sapphire laser.

Positive and negative controls for collagen type II and collagen type IV were performed on paraffin sections of human kidney and bovine mammary gland, respectively. This research adhered to the tenets of the Declaration of Helsinki.

Results

Posterior Lens Capsules of Children Presenting with Congenital Unilateral Posterior Cataract

Capsules were collected from 3 children who had surgery for congenital unilateral posterior cataract and presented with a posterior capsule plaque after lens removal. Clinically, an abnormal vitreolenticular interface was found on attempting posterior capsulorhexis; however, no signs of PFV were observed. Child A (boy) and child B (girl) were 3 months old at the time of surgery; child C (boy) had surgery at the age of 11 years. All 3 children had a normal gestation, birth, and development. There was no family history of cataract in any of these children. In child C, a large part of the posterior capsule plaque was removed before posterior capsulorhexis was performed. All specimens were collected and processed according to the procedure described previously (Figures 2 and 3).

Figure 2
Figure 2:
Immunohistochemistry of posterior capsule plaques and capsules in children with congenital unilateral posterior cataract. A to C: Collagen type IV highlighted with fluorescein red (arrows). A’ to C’: Collagen type II highlighted with fluorescein green. Arrows indicate collagen type II adhering on the posterior surface of the posterior lens capsule; asterisks indicate collagen type II in the posterior capsule plaques.
Figure 3
Figure 3:
Immunohistochemistry of posterior capsule plaques and capsules in children with congenital unilateral posterior cataract (A and B). Both collagen type IV (red) and collagen type II (green) are highlighted simultaneously.

Indirect immunohistochemical analysis of the specimens showed the presence of collagen type IV in all 3 posterior lens capsules. Collagen type II was found to adhere tightly to the posterior surface of the posterior lens capsule in all 3 specimens. Collagen type II also was found in all posterior capsule plaques. In child C, no cell nuclei were found in the posterior capsule plaque after DAPI staining.

Posterior Lens Capsules of Children and Adult Controls

Three posterior capsule specimens were collected from 3 children who had been operated on for bilateral congenital cataract and in whom no posterior capsule plaque was observed. In all 3 children, the posterior capsule and the anterior hyaloid membrane had been easily viscodissected (Figure 4). Child A (girl) was 2 years 11 months old at the time of surgery. Child B (girl) was operated on at the age of 6 years 6 months. Child C (boy) was 4 years 3 months old at the time of surgery. In addition, 3 posterior capsule specimens were collected from adult patients who had been operated on for senile cataract and also presented without a posterior capsule plaque and with a normal Berger space (Figure 5). Adults A (woman) and adult B (man) were operated on at the age of 71 years and adult C (woman) at the age of 64 years. None of the 3 adults had a significant medical or ophthalmologic history. All specimens were collected and processed according to the procedure described earlier.

Figure 4
Figure 4:
Immunohistochemistry of posterior capsules in pediatric cataracts with normal vitreolenticular interface. A to C: Collagen type IV highlighted with fluorescein red (arrows). A’ to C’: Collagen type II highlighted with fluorescein green (absent).
Figure 5
Figure 5:
Immunohistochemistry of posterior capsules in senile cataracts with normal vitreolenticular interface. A to C: Collagen type IV highlighted with fluorescein red (arrows). A’ to C’: Collagen type II highlighted with fluorescein green (absent).

Indirect immunohistochemical analysis showed collagen type IV in all 6 posterior lens capsules, whereas none of the specimens contained collagen type II. No cell nuclei were found either, except in 1 child.

Discussion

This article focuses on the vitreolenticular interface in congenital unilateral cataract patients presenting with a posterior capsule plaque but not showing any features of PFV. Posterior capsule plaques might be associated with any other type of lens layer opacification. In a study by Praveen et al.,3 70 of the 90 eyes with posterior capsule plaques presented with a complete white cataract. Forty-five cases of nuclear lens opacities and 16 of the 21 cases of cortical lens opacities were found to be associated with posterior capsule plaque in a study performed by the IATS group.5 These authors also observed that cataract density was frequently increased in the posterior part of the fetal nucleus and the cortex nearer the posterior capsule when unilateral congenital cataracts were associated with a posterior capsule plaque.5 Praveen et al.3 further observed posterior capsule plaque in 90 (13%) of 670 eyes of children having unilateral or bilateral cataract surgery between 1 month and 15 years of age. A study by Morrison et al.15 found a much lower incidence of posterior capsule plaque in bilateral congenital cataract cases compared with unilateral cases, and a video-documented assessment performed by the IATS group5 showed posterior capsule plaque in 73 (88%) of 83 eyes with monocular infantile cataract. These results suggest that posterior capsule plaques are more common in unilateral infantile cataract cases, leading to the hypothesis that unilateral cataracts with posterior capsule plaque are caused by PFV, generally a unilateral condition, even if no obvious hyaloid remnant is visible.16

Posterior capsule plaques are difficult to peel without rupturing the posterior lens capsule and are often indicative of the presence of an abnormal vitreolenticular interface. These changes in Berger space can be appreciated only after a posterior capsulorhexis involving viscodissection of the posterior capsule and the anterior hyaloid membrane. They will be missed when the posterior capsule and anterior hyaloid are removed simultaneously using a vitrectome. However, these adhesions make the surgical procedure of posterior capsulorhexis more challenging and sometimes require unintended anterior vitrectomy.10 In a series by Praveen et al.,3 the posterior capsule plaque was peeled in 41 eyes, anterior vitrectomy was performed in 41 eyes, and in-the-bag IOL positioning was possible in only 54 of the 90 eyes. In our experience using bag-in-the-lens IOL implantation, rupture of the anterior hyaloid membrane is inevitable in cases with strong vitreolenticular adherence. However, in the case of a meticulously performed viscodissection before the posterior capsulorhexis and because of the solid nature of the vitreous in these young children, vitreous prolapse does not always occur and anterior vitrectomy is not always indicated.

Haargaard et al.17 found PFV, defined as the presence of a retrolenticular fibrovascular membrane or persistent hyaloid stalk and traction on the ciliary processes, in 56 (57%) of 99 eyes of children between 0 years and 17 years presenting with unilateral cataract. Forster et al.,18 on the other hand, report a PFV prevalence of 20%. The IATS group5 observed PFV in 18 (22%) of 83 eyes, although they excluded severe cases. These findings allow us to conclude that there is still a large group of unilateral infantile posterior cataracts without evident signs of PFV.

Using a broader definition of PFV, Müller-Eidenböck et al.4 found that all eyes with congenital unilateral cataract (31/31) showed signs of PFV. They added findings of minimal fetal vascular remnants, including nonperfused spidery ghost vessels within the posterior capsule plaque, an abnormally thickened hyaloid face, or a membrane-like structure continuous with the posterior capsule lens opacity. As such, they argued that the posterior capsule plaques and associated opacities observed in the nucleus and cortex of infants with unilateral cataract might occur as a result of a mild form of PFV and that the posterior lens capsule and anterior hyaloid face are invaded by fetal vessels, resulting in an abnormally strong attachment site between the posterior capsule, cortex, and nucleus before dissipating.4

The present histologic study supports the hypothesis that abnormal formation of the vitreolenticular interface is probably the trigger for many infantile cataracts. Histologically, the posterior lens capsule is a basement membrane of the lens, mainly formed by a network of laminin and collagen type IV.11 Nishi et al.12 did not find any collagen type II when studying the types of collagen synthesized by lens epithelial cells (LECs) in human cataract. Although Savontaus et al.19 reported type II collagen mRNA in the embryonic lens of mice, collagen type II was never shown in a human lens. The collagen found in the vitreous body is mainly type II (>75%) but never type IV.13 Our finding of type II collagen adhering to the center of the posterior lens capsules in all 3 developmental cataract samples is indicative of dysgenesis of Berger space.

By using indirect immunohistochemistry, the specificity of staining collagen type II and collagen type IV increases. Samples of patients and controls were collected and processed at different times, raising potential issues in comparing the staining intensity of these samples. To minimize these issues, a fixed protocol was used for sample fixation, preparation, and photographing. When processing a sample, new positive and negative controls for collagen type II and IV were also performed (on paraffin sections of the human kidney and bovine mammary gland, respectively), decreasing the risk for false negative and false positive results.

Three samples were included in every subgroup of this study: 3 children with congenital unilateral posterior cataract, 3 children with bilateral congenital cataract (controls), and 3 adults with senile cataract (controls). These small samples are mainly because cataract in children is a rare pathology and does not present that often for surgery.1 The posterior capsule samples extracted during the posterior capsulorhexis procedure were also small (5.0 mm in diameter) and fragile. Collecting, fixating, and processing these samples can be challenging. The planning of a larger multicenter study is indicated to strengthen the findings in this study.

The pathophysiologic pathway inducing cataract formation in the case of an abnormal vitreolenticular interface is still a matter of debate. Müller-Eidenböck et al.4 hypothesized that posterior capsule plaque and cataract formation are caused by vessel invasion from PFV through the posterior lens capsule. Others believe that posterior capsule plaque and vitreolenticular adhesion are a form of cicatrization, with hyalocytes and/or equatorial LECs healing a defect in the posterior lens capsule. These theories are supported by the difficulty in peeling the posterior capsule plaque from the posterior capsule and the typical adhesion of the anterior hyaloid membrane underneath the plaque. They may explain the presence of collagen type II inside the posterior capsule plaques found in this study. On the other hand, no discontinuity of the posterior lens capsules was observed underneath the plaques. Another theory might be that the lack of separation between the posterior capsule and the anterior hyaloid membrane induces mechanical and environmental changes at the level of the posterior capsule, which influences the migration and differentiation of normal LECs and lens fibers. This would explain the need for the formation of Berger space in normal developing human eyes; posterior capsule plaques would then originate from lens epithelial–mesenchymal transition. However, this process is associated with increased expression of extracellular matrix proteins (including collagen type I and III), smooth-muscle actin, and intermediate filaments; it never involves collagen type II.12,20

In conclusion, using indirect immunohistochemical labeling, this study found that the anterior hyaloid membrane (containing collagen type II) firmly adhered to the removed posterior lens capsule and was indicative of the dysgenesis of Berger space in congenital unilateral posterior cataract. The posterior capsule plaques typically associated with this type of cataract contain collagen type II, a type of collagen that has never been seen in a human lens to date. Our results are strengthening the belief that these congenital unilateral cataracts with posterior capsule plaque are likely to be a reflection of some developmental anomalies in the anterior vitreous face. Further study would be necessary to validate the idea that these cataracts are truly a specific subgroup of congenital cataract and arise secondary to a dysgenesis of the vitreolenticular interface. If so, the term anterior vitreolenticular interface dysgenesis might be appropriate.

We believe that further knowledge of the biochemical composition of posterior capsule plaque and posterior capsules in these cataracts using proteomic approaches would be beneficial to understand cataract formation in children and ultimately would improve the surgical outcomes of cataract surgery in these particular cases, which are generally characterized by a poor prognosis.

What Was Known

  • Congenital unilateral cataract is often associated with posterior capsule plaque and challenging cataract surgery. Little is known about its underlying pathophysiology.

What This Paper Adds

  • The adherence of collagen type II to the posterior surface of the posterior lens capsule supports the hypothesis that congenital unilateral cataract with a posterior capsule plaque hints at an abnormality at the vitreolenticular interface.

References

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