Multimodal imaging of a micro-anatomical structure in the vitreous base

The vitreous base (VB) is a region that extends approximately 1.5–2 mm anteriorly from the ora serrata (OS) on the pars plana and 3–5 mm posteriorly behind the OS on the retina; the posterior extension of the VB may enlarge with age [1‐3]. The VB is involved in various vitreoretinal diseases [4‐7]. However, some mechanisms in these diseases cannot be explained due to lack of the ultrastructure knowledge related to the VB.

Clinically, progression of newly diagnosed rhegmatogenous retinal detachment (RD) ceases on the posterior edge of the VB (PEVB), whereas in traumatic cases or selected long-standing RD cases with proliferative vitreoretinopathy (PVR), OS and ciliary epithelium detachment (Fig. 1) may occur along with RD [8‐10]. However, it remains unclear why some cases of RD do not continue beyond the PEVB or how the OS detaches from its underlying tissue. Meanwhile, we have also observed that torn pigment tissues from the underlying pars plana/choroidal substantia adhere firmly to the separated VB in blunt eyeball injuries with VB avulsion (Fig. 1B), and a whitish background in the pigment-denuded region with embedded choroidal vessels is often observed (Fig. 1D). It has been reported that the strong adhesion is caused by VB fibrils passing through the crypts of the peripheral retinal surface and blending with the lining basement membrane or with the plasmalemma of Müller cells [11]. However, current anatomical knowledge cannot clearly explain how large-scale pigmented sheet is torn from the underlying tissue.

Following observations of the VB micro-anatomical structure, we collected a series of peripheral retina images during vitrectomy surgery and compared these with images of ruptured VB tissue and healthy donor eyes obtained from microscopic observation. The ultrastructure located in the VB is fully visualized, and clinical VB abnormalities related to RD and trauma can be explained by the micro-anatomical characteristics of this structure. The findings of this study may help surgeons identify pathological signs and make rational decisions during surgery.

This is a study of practical anatomy. The objects of the study are categorized into three groups: 1) Fundus images, which were obtained from 4 patients during vitrectomy surgery, included 2 with RD and 2 with blunt eyeball injuries; the images from 2 patients with blunt eyeball injuries were both taken from the avulsed VB region (Fig. 1A and D). The four fundus images were selected based on the corresponding anatomical characteristics of the VB, and all these four images were captured during the surgery through RESIGHT viewing system attached in OPMI LUMERA 700 Zeiss surgical microscope. The sclera depressing was introduced during surgery to expose the VB region clearly in all cases. 2) After locating the vitreous base abnormalities intraoperatively, light microscopic observations of vitreous base were applied on two specimens (Fig. 2A and B) from one eye (patient 1) post-trauma (Fig. 1A) and one healthy eye post-mortem, the purpose of the light microscopic observation is to know the cellular component of the VB specimens; and then 3) Transmission electron microscopic images of the specimen,which was captured from the other eye (patient 2) post-trauma during the vitrectomy surgery (Fig. 1D), were analyzed to understand the spatial relationship among different cells and tissues within the VB region.

Two samples containing detached ciliary epithelium and peripheral retina along with the avulsed VB region were both extracted from patient 1 and patient 2 during the surgery. The specimen from patient 1 was immediately placed on a cellulose acetate membrane during surgery (Fig. 1B subset) and embedded in 10% paraformalin for light microscopic observation under hematoxylin and eosin and periodic acid-Schiff staining (Fig. 2A). The specimen from patient 2 was embedded in 2% glutaraldehyde for transmission electron microscopic observation (Fig. 3). The post-mortem healthy eye was stained with Masson stain for microscopic observation (Figs. 2B and C).

The surgical microscope used for operation was OPMI LUMERA 700 ZEISS. The microscope for histopathology observation was Olympus BX53A with U-LHLEDC. The transmission electron microscope was JEM-1230,JEOL.

Before surgery, we had planned to obtain the samples containing detached ciliary epithelium and peripheral retina within the avulsed VB region caused by trauma if the detached tissue cannot be re-attached during the revised surgery for patient 1 and patient 2. The patients were notified of our plan before surgery, and consent was obtained. This study was approved by the Institutional Review Board of Peking University Third Hospital and was conducted in accordance with the tenets of the Declaration of Helsinki.

By combining the microscopic findings and fundus images obtained during vitrectomy surgery, we demonstrated the location of the CB-C-R connector in the VB, which is composed of dense collagen fibers that are different from vitreous fibers. The spatial relationship between the connector and its surrounding tissues were also defined (Fig. 4). The important anatomical markers related to the CB-C-R connector are the PEVB, VBI, and OS, which were all demonstrated clearly by both histological and clinical observations.

The reasons of choosing light microscopy and transmission electron microscopy are as follows: 1) It is a mandatory procedure to observe the samples obtained from patients during surgery for histopathological diagnosis. 2) The transmission electron microscopic observation can demonstrate clearly the cellular content of CB-C-R as well as their spatial relationship with adherent tissues in the VB region. 3) Due to the limited sample size and numbers of specimen, it would be hard to choose other modalities (immunohistochemistry staining or scanning electron microscopic observation) for histopathologic observation to demonstrate the structure of CB-C-R.

Ciliary epithelium tears and OS dialysis, which break the connection between the CB-C-R connector with its surrounding tissues including the choroid, OS, ciliary epithelium, and VB, may occur following VB avulsion. Feng [12] reported that in 10 ruptured eyeball injuries, choroidal rupture occurred posterior to the pars plana in all cases. The location of the CB-C-R connector was consistent with the locations of choroidal rupture, which may be explained by the CB-C-R connector being pulled away vertically from the choroid towards the vitreous cavity by the VBI and lead to choroidal rupture.

The different boundaries of RD are associated with the different compromised connections among different tissues within the VB. 1) RD ceases posterior to the PEVB (Fig. 5A). Histopathologically, the VBI anterior to the PEVB allows the VB to be firmly attached to the retina (Fig. 3A circle and 3B star). Meanwhile, the connection between the retina and the underlying retinal pigment epithelium layer is stronger in the anterior PEVB than in the posterior PEVB. Although we did not obtain images of the anatomical connection between the retina and retinal pigment epithelium layer in the VB, we observed that the retinal pigment epithelium sheet was attached to the detached retina, leaving a large retinal pigment epithelium-denuded region next to the OS, as shown in the fundus image of patient 2 (Fig. 1D). 2) RD extends beyond the PEVB and cease at the OS where the CB-C-R is located (Figs. 5D and 5E). As PVR progresses into the circumferential type (grade C) that leads to centrepedal-contracted RD, the VBI in the OS is the only site where vitreous fibers are connected to the pigment epithelium layer [13] (Fig. 5E). The CB-C-R connector functions as a fixed and firm plug that resists the pulling force of PVR. 3) In cases of trauma and long-standing RD with severe PVR, the RD perimeter extends beyond the OS and involves the ciliary epithelium (Figs. 1 and 5D). Clinically, OS avulsion, RD, and ciliary epithelium detachment were observed. Histopathologically, the CB-C-R connector separated from the underlying choroidal tissue and pigment/non-pigment epithelium along with RD, which exposed the uveal tissue to the vitreous cavity.

Although the techniques and instruments have been improved dramatically, circular scleral buckle procedure after vitrectomy still plays an important role in preventing retinal tears posterior to the PEVB in eyes undergoing long surgeries or multiple posterior vitreous detachment (PVD) [14‐17]. Variations of the VBI, which may cause complications [3, 18], may also be prevented by circular scleral buckle surgery. When PVD occurs, the peripheral retina posterior to the PEVB is most vulnerable to the tangential pulling force from the VBI due to its connection to the vitreous fibers and peripheral retina. Scleral buckle procedures reduce vitreous traction to the retina and strengthens the stability of the transition zone between regions of PVD and non-PVD posterior to the PEVB.

The number of samples in this study was limited. Additionally, some samples were obtained from eyes post-trauma, which may not reflect normal VB status. Hence, any conclusions drawn from the study should be taken with caution.