Ultrastructure of Anterior Lens Capsule and Epithelium in High-Myopic Cataracts


 BACKGROUND: The mechanism of high-myopic cataract (HMC) is not fully understood yet. The ultrastructure of anterior lens capsule (aLC) and lens epithelial cells (LECs) may provide some clues to this issue.METHODS: Capsulorhexis samples from 10 eyes with HMC and 10 eyes with age-related cataract (ARC) were analyzed for morphologic characteristics using transmission electron microscopy (TEM).RESULTS: Several common pathologic disorders of LECs, including pleomorphic cellular structure, extensive intracellular vacuoles, swollen organelles, degeneration and necrotic lesions, could be observed in both HMC group and ARC group, to a similar extent. Specimens of HMC group disclosed remarkable extracellular spaces, interdigitation of LECs as well as protuberances of aLC towards LECs. CONCLUSIONS: The ultrastructure of aLC and LECs in HMC demonstrated some specific pathologic disorders in comparison with ARC, which might play a role in the formation of cataract in high myopes.


Background
There is an unprecedented epidemic of short-sightedness in many parts of the world, especially in East Asia [1]. Myopia and high myopia are predicted to impact 49.8% and 9.8% of global population respectively by 2050, emerging as an incremental worldwide public health issue [2]. This phenomenon foreshadows a considerable increase in complications of pathologic myopia, for example, the highmyopic cataract (HMC). Relationship between cataract (posterior subcapsular cataract and nuclear cataract) and high myopia is well established [3]. Nevertheless, mechanism of HMC is not completely understood yet.
It is well known that anterior lens capsule (aLC) and lens epithelial cells (LECs) not only play a physical defense role, but also serve as a regulatory barrier between aqueous humor and lens bers to guarantee the transparency of crystalline lens [4,5]. We performed morphology analysis of aLCs and LECs obtained from routine cataract surgeries by transmission electron microscope (TEM) to further investigate the underlying etiology of HMC. To the best of our knowledge, no research has focused on this topic so far.

Patients
This electron microscopic investigation was conducted in Fujian Provincial Hospital and Fujian Medical University. Twenty consecutive patients diagnosed as ARC (10 patients) or HMC (10 patients) were recruited. We included ultra-high myopes whose axial length exceeding 29mm. The preoperative best corrected vision acuity was worse than 0.4 logMAR and cataract surgical intervention was required. Every patient underwent a detailed history inquiry and a comprehensive ophthalmic examination (Table 1). Exclusion criteria included: (1) systemic diseases such as diabetes mellitus and hypertension; (2) ocular disorders such as ocular infection, uveitis, glaucoma, and retinopathy; (3) history of ocular trauma or intraocular surgery; (4) any form of steroid usage. Written informed consents to participate in this study were obtained from all patients before the operation.

Results
Ten patients with HMC (aged 61.70 ± 11.14 years, axial length: 30.54 ± 0.79 mm) and ten patients with ARC (aged 66.7 ± 5.69 years, axial length: 23.22 ± 0.62 mm) were recruited in this study from July 2019 to June 2020 (shown in Table 1). We were not able to associate patients' age or nuclear density with TEM pathologic changes due to small sample size.
Relatively normal segments of aLC and LECs were occasionally observed in ARC group, which consisted of homogenous-appearing basement membrane and regular single-layered cobble-shaped cells with round nuclei, in absence of degeneration or necrosis (shown in Fig. 1). By contrast, the relatively normal architecture was not observed in HMC group.
Several pathological alterations could be seen in both HMC group and ARC group, to a similar extent: (1) LECs were altered from cuboidal shape to pleomorphic shape, with extensive intracellular vacuolization of cytoplasm and edema of mitochondria, endoplasmic reticulum as well as other unde ned organelles (shown in Fig. 2a, 2b, 2c). (2) There existed degeneration and necrotic lesions of LECs: the degenerated cells, with remarkable aforementioned pathological changes, showed much lower electron density than the normal ones (shown in Fig. 2b); the necrotic epitheliums possessed ruptured cytomembrane and swollen nonfunctional organelles (shown in Fig. 3). Besides, apoptosis was discovered in neither HMC group nor ARC group. (3) Less frequent disorders comprised an increase of lysosomes inside some LECs and evidently attened LECs with heavy-dyed nuclei (shown in Fig. 2b, 2d).
According to our observation, specimens of HMC group disclosed more obvious extracellular vacuolization, placed between the lateral margins of adjacent LECs or between the basal lamina and LECs, varying from less than 1µm to over 10µm (shown in Fig. 4). Location of extracellular spaces varied among different specimens. In Patient 12, 13, 16, 17 and 20, most of the spaces were distributed close to epithelium-basal lamina border (shown in Fig. 4a). In Patient 15,18, they were mostly located close to the apical surface (shown in Fig. 4b). While in Patient 11,14,19, these two distribution manners could both be seen. A great portion of the extracellular spaces in HMC specimens was embedded with debris materials, 0.1-1µm in diameter (shown in Fig. 4b, 4d). Some were amorphous, some contained cristae-like fragments or microgranules and others possessed visible outer membrane. In some sections of the HMC specimens, concomitant with the extracellular vacuolization were multiple cell membrane folds, interdigitating with neighboring LECs (shown in Fig. 4c, 4d). The interdigitation could seldom be seen in ARC group. In addition, some LECs in HMC group showed branched cytoplasm which resembled broblasts (shown in Fig. 4a, 5a).
Another predominant disorder observed in ve HMC specimens (Patient 12, 13, 15, 17, 18, aged 48, 52, 59, 68 and 71 respectively) was protuberances of basement membrane towards LECs. These protuberances were in different morphologic types, ranging from minimal bulge less than 1µm to long protuberance over 20µm (shown in Fig. 5). And there existed large extracellular spaces and debris around the polypoid protuberance. This disorder was obvious in Patient 13 and Patient 18, which was widespread over the whole basement membrane. In Patient 13, the electron density of some polypoid protuberances was much lower than normal basement membrane. A relatively transparent linear lesion extended from the polypoid protuberance towards anterior chamber in a vertical (shown in Fig. 5a, 5b, 5c). What's more, some segments of basal lamina in HMC group showed parallel laminal rare cation (shown in Fig. 6).

Discussion
In this study, we investigated the ultrastructure of aLC and LECs in HMC compared with ARC by TEM, to analyze the possible etiological mechanism of HMC. A lot of efforts have been devoted to explore the electron microscopic architecture of lens capsule since decades ago [6-15]. However, high-myopic cataracts haven't been involved so far.
A sheet of epitheliums is located between anterior lens capsule and ber cells, with region-speci c features [4,16,17]. Though the central epitheliums we collected from routine phacoemulsi cation surgery are considered essentially quiescent in terms of cell cycle, they drive active metabolic and transporting machinery to guarantee the homeostasis of the whole crystallin lens [4,[16][17][18][19]. By TEM, we discovered some common pathologic features in both HMC group and ARC group, to a similar extent, such as pleomorphic cellular structure, vacuolated cytoplasm, swollen organelles, as well as degeneration and necrotic lesions, which were also con rmed by previous studies of other cataractic types Previous studies also demonstrated that with aging, the deposit of oxidized lens constituents together with the impairment of antioxidative function (which LECs play a role in) can bring about opacities in crystallin lens, resulting in caractogenesis [20][21][22][23][24]. Any disruption of morphologic structure might be implicated in dysfunction of LECs, which would accelerate cataract formation.
As to the underlying etiology of HMC, one of the possible mechanisms might be that the crystallin lens of high myope suffers from higher concentration of oxygen, which is resulted from impaired physical and chemical barriers of vitreous body [3,[24][25][26]. As a consequence, the surrounding ROS of crystallin lens add to the oxidative damage to LECs as well as the risk of cataract formation in high myopic eyes. What's more, a number of signi cantly changed metabolites in aqueous humor (where the anterior lens capsule was bathed in directly) of high myopes were identi ed by Ji YH et al [27], but it is not veri ed yet whether or not the metabolic variations are involved in the morphological changes of LECs in high myopes.
In our study, there exist more extensive extracellular spaces and sometimes interdigitation between LECs in HMC group (shown in Fig. 4, 5). Similar extracellular vacuolization was also reported in a TEM study of LECs in presenile cataract [13]. Since all specimens in our experiment went through the same surgical procedure, we consider that the greater extracellular spaces in HMC group are irrelevant to the surgical stimulation. We proposed that the larger extracellular spaces might be the consequence of cell contraction and cytoskeletal reorganization. Firstly, an inverted microscopic research by Andjelic, et al demonstrated that LECs possessed the ability of contraction, and under non-speci c stimulation, the intercellular width changed from barely noticeable to several microns [28]. Furthermore, larger extracellular spaces were also observed in LECs of a transgenic mouse model expressing Rho GTPase inhibitor compared with wild-type mouse LECs under TEM, while Rho GTPase was considered as a crucial point in actin cytoskeleton arrangement [29][30][31]. Researchers have also suggested that an inappropriate accumulation of transforming growth factor-β (TGF-β) induced remodeling of lens cytoskeleton through Rho family of GTPases [30,31]. In the meanwhile, Zhu XJ and his copartners found signi cant elevated TGF-β2 concentration in the aqueous humor of highly myopic eyes, in proportion to axial length [32]. Therefore, it might be worthwhile to further explore the expression of TGF-β and Rho GTPase in surgical capsule samples of HMC as well as their association with lens epithelial cytoskeleton. In addition, the interdigitation between LECs was also observed in cataracts with retinal pigmentosa and uveitis [7,9]. Andjelic, et al also reported similar interdigitation between adjacent LECs at the basal side but they were not related to the type of cataract (the speci c cataract types were not provided though) [6]. However, in our study, the interdigitation of LECs were rarely found in ARC group.
Another signi cant characteristic observed in ve high-myopic specimens (Patient 12, 13, 15, 17, 18, aged 48, 52, 59, 68 and 71 respectively) is the protuberances of extracellular matrix (ECM) towards LECs (shown in Fig. 5). This phenomenon was not reported by Andjelic and colleagues, whose experiment focused on the LECs attachment to aLC in various cataract types by a combination of TEM, scanning electron microscopy and confocal microscopy [6]. However, Hawlina M et al discovered similar protuberances with degraded cellular materials inside at the epithelium-basal lamina border in 86.6% samples of intumescent cataract and in 34.3% samples of nuclear cataract [33]. In their opinion, the function of the protuberances was to capture degrading LECs debris and after that, new layer of epithelium would be deposited upon the gap space. Different from this study, we didn't nd captured cellular debris in the protuberances, and we observed distorted epithelium with notable extracellular spaces upon the protuberances rather than newborn LECs (shown in Fig. 5). Richiert DM, et al and Zhang XH, et al respectively observed elevated secretion of ECM components ( bronectin and laminin) in animal model LECs treated by TGF-β, which was proved to increase in aqueous humor of highly myopic eyes [34][35][36].Hence, the speci c components and origin of the protuberances along epithelium-basal lamina border in HMC need further investigation.
There are several limitations in this study: the crucial ones are the small sample size and the qualitative description; what's more, our hypothesis about the etiology of extracellular spaces among LECs and protuberances in aLC cannot be merely established by morphologic observation, so immunohistochemistry and other con rmable methods are needed. Therefore, our next step will be expanding sample size and nding objective evidence to explain our observation of lens capsule in HMC.

Conclusions
Compared with ARC, the ultrastructure of aLC and LECs in HMC demonstrated some speci c pathologic disorders, which might play a role in the formation of cataract in high myopes. As far as we know, no research has explored the electron microscopic structure of aLC and LECs in HMC yet.

Availability of data and materials
The data and materials generated and analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by Grant No.2019J01187 from Natural Science Foundation of Fujian Province, China.

Authors' Contributions
WJW, YBW and YJC designed the investigation; the surgeries were conducted by WJW. XRZ and MXW conducted the experiment; MTY, XRZ and MXW analyzed and interpreted the results; the paper was drafted by YBW and revised by WJW. All authors read and approved the nal manuscript.  basal membrane (anterior lens capsule); iv, intracellular vacuole; arrow: mitochondrion; asterisk: lysosome. b. n, nucleus; bm, basal membrane (anterior lens capsule); arrow: mitochondrion; asterisk: lysosome; rectangle: endoplasmic reticulum. c. bm, basal membrane (anterior lens capsule); iv, intracellular vacuole.   Protuberances of basal membrane, surrounded by pathologic extracellular spaces and debris materials in a 52-year-old male patient with HMC ( Fig. 5a-5c: Patient 13). A relatively transparent zone extends from the polypoid protuberance in a vertical way (indicated by an ellipse). a. es, extracellular space; p: protuberance; star: broblast-like epithelium; asterisk: branched cytoplasm of broblast-like epithelium. b.

Figure 6
Segments of aLC in HMC group with parallel laminal rare cation (Patient 18).