Purpose
To investigate long-term ultrastructural changes in the retina after internal limiting membrane (ILM) peeling through the examination of morphologic changes 3 years after vitrectomy in cynomolgus monkeys.
Design
Laboratory investigation.
Methods
Pars plana vitrectomy was performed, followed by ILM peeling, in 2 primate eyes. Ultrastructural changes were investigated using light microscopy and transmission and scanning electron microscopy 3 years after ILM peeling.
Results
The remaining posterior vitreous and ILM-peeled areas were clearly recognized after the long-term follow-up. The exposed Müller cell processes were partially damaged, while regenerative spindle-shaped Müller cell processes developed, covering most of the retina. Notably, the nerve fiber layer was found to be uncovered and exposed to the vitreous space owing to misdirection of glial wound healing in some parts. In these areas, glial wound healing occurred beneath the nerve fiber layer. Although the glial cells covered the damaged areas, there was no apparent ILM regeneration in the shape of a continuous flat sheet, with the exception of accumulated deposits of basement membrane materials.
Conclusions
Although the retinal structures were well preserved after ILM peeling, ILM peeling resulted in mild damage to the vitreoretinal interface, which was not completely restored even after 3 years. The multilinear shape of the exposed nerve fiber may explain the previously reported dissociated optic nerve fiber layer appearance. The glial cells produced basement membrane materials around their processes, although they did not restore the ILM as a flat sheet.
The pathogenesis of various vitreoretinal diseases is still not fully understood. However, there is agreement regarding the important role of vitreous attachment and vitreous traction to the underlying internal limiting membrane (ILM) and retina. The ILM is originally produced by Müller glial cells during the development of the retina and provides basement membrane under normal conditions or extracellular matrix for cellular migration and contraction under pathologic conditions. Histologic examinations of excised ILMs have demonstrated that migrating cells of various origins are located on the ILM and that these cells are associated with collagen fibers of various diameters. ILM peeling during vitrectomy drastically increases the closure rate in macular holes. ILM peeling allows for the complete removal of the posterior vitreous cortex, contraction of the extracellular matrix, and cellular migration. In addition, ILM peeling has been widely performed to treat patients with vision-threatening vitreoretinal diseases, such as severe myopic retinal detachment or proliferative vitreoretinopathy.
However, there has been some concern regarding the adverse effects of this technique, since the ILM constitutes the basement membrane of Müller cells and the inner barrier of the neural retina. In our previous report, damaged underlying Müller cell processes were observed just beneath the ILM immediately after ILM peeling. In some areas, glial cell processes laying between the ILM and nerve fiber layer were removed. Consequently, the nerve fibers were exposed directly to the vitreous space without glial cells. Although we previously reported the early morphologic changes observed after experimental ILM peeling in cynomolgus monkeys, the long-term outcomes after ILM peeling remain elusive. Although morphologic changes after ILM peeling have been observed under optical coherence tomography (OCT), the correlations between OCT findings and pathologic changes are clinically important. The irregular appearance of the retina after ILM peeling surgery in the fundus or OCT examinations may reflect pathologic changes. In this report, we evaluated ultrastructural changes and remodeling of the retina 3 years after experimental vitrectomy with ILM peeling.
Methods
Animals
The study was designed as a laboratory investigation. All experiments were performed in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the animal care and use committee at Kyushu University, Fukuoka, Japan. Two eyes from 2 cynomolgus monkeys between 3 and 4 years of age were used in this experiment. The cynomolgus monkeys were restrained in a squeeze cage and injected intramuscularly in the thigh with 20 mg/kg of ketamine hydrochloride (Sankyo Yell Pharmaceutical Products Co, Ltd, Tokyo, Japan) for general anesthesia. The monkeys were subsequently transported to the operating room.
Vitrectomy and Internal Limiting Membrane Peeling
Standard 3-port pars plana vitrectomy and ILM peeling were performed in a closely similar fashion to that carried out in human patients, as previously reported. The internal limiting membrane was stained a light green color with 0.5% indocyanine green (ICG) solution. The internal limiting membrane was removed in a circular fashion up to the temporal arcade and optic disc. The instruments were then removed and the sclerotomy ports were closed using 7-0 polyglactin sutures.
The monkeys were euthanized with an overdose of sodium pentobarbital (200 mg/kg) and the eyes (n = 2) were enucleated 3 years after surgery. The enucleated eyes were fixed with 1% glutaraldehyde and 2% osmium tetroxide. The posterior parts of the eyes were prepared under a biomicroscope (Nikon, Tokyo, Japan) for the histopathologic analyses.
Transmission Electron Microscopy
The posterior segments were fixed in 1% glutaraldehyde and 1% paraformaldehyde in phosphate-buffered saline. The retinas were postfixed in veronal acetate buffer osmium tetroxide (2%), dehydrated in a series of ethanol and water, and embedded in Epon (Electron Microscopy Sciences, Hatfield, Pennsylvania). Semi-thin sections were cut from the block and observed under a light microscope (Olympus, Tokyo, Japan). Ultrathin sections were mounted on copper grids and observed with a JEM 100CX (JEOL, Tokyo, Japan) and H-7650 and H-7700 electron microscope (Hitachi, Tokyo, Japan).
Scanning Electron Microscopy
The retinas were postfixed in veronal acetate buffer osmium tetroxide (2%) and dehydrated in ethanol and water. The retinas were saturated in t-butyl alcohol, and critical point drying was performed. The tissue was then placed on stubs by means of self-adhering carbon tabs and sputtered with Au of 20 nm thickness using an argon plasma coater. The retinal surface of the eyes was then studied under a scanning electron microscope (JSM-840A; JEOL, Tokyo, Japan).
Results
Biomicroscopy and Light Microscopy
The fixed eyes were cut and the posterior part was observed under a biomicroscope ( Figure 1 ). The eyecup showed a well-preserved structure in the posterior portion of the monkey eye, similar to that observed in the human eye ( Figure 1 , Top left). While the optic disc and macula were clearly recognized, the area with ILM peeling was not obvious under wet conditions ( Figure 1 , Top left). The sample preparation using critical point drying minimized ultrastructural changes for successful scanning electron microscopy ( Figure 1 , Top right). Under dry conditions, the artificial posterior vitreous detachment and remaining posterior vitreous were clearly observed around the posterior part of the eyes. Light microscopic images of semi-thin sections showed well-preserved structures of the retina in the ILM-peeled areas compared to those noted in the neighboring normal retina. The retinal thickness of the ILM-peeled areas was similar to that of the normal areas ( Figure 1 , Bottom left and Bottom right).
Scanning Electron Microscopy
The low-magnification images provided a bird’s-eye view of the posterior parts of the eye and the major structures, such as the remaining posterior vitreous, ILM, macula, and optic disc ( Figure 2 , Top). The remaining posterior vitreous was rolled up on the vitreous surface ( Figure 2 , Middle left). The normal area without ILM peeling was observed as a dark area on the retina, while the lighter area that surrounds the foveal pit is the zone of ILM detachment. The retina with ILM peeling exhibited an irregular surface structure with cellular components, compared to the smooth flat surface structure of the normal retina ( Figure 2 , Top). Most parts of the retina with ILM peeling were covered with Müller cell processes, whereas glial cell processes reproduced the flattened surface of the retina ( Figure 2 , Middle right). In some parts, multilinear wave fibrillary structures considered to be denuded nerve fibers were observed without covering Müller cell processes ( Figure 2 , Bottom left and Bottom right, arrows). The denuded nerve fibers created a rough retinal surface without glial processes. There was no apparent ILM regeneration as a flat sheet covering the ILM-peeled retina.
Transmission Electron Microscopy
The regenerative Müller cell processes extended and covered the wounded areas with ILM peeling ( Figure 3 , Top). The abundant intermediate filaments in the cytoplasm of these cells indicated that these cells were Müller glial cells. The surface of the exposed retina was covered with densely packed, multilayered Müller cell processes ( Figure 3 , Middle left). In some areas, the nerve fiber layer was uncovered and exposed to the vitreous space owing to misdirection of glial wound healing ( Figure 3 , Middle right). The glial wound healing developed beneath the nerve fiber layer or shaped the Müller cell wound cleft ( Figure 3 , Middle right and Bottom left). In the Müller cell wound cleft, glial cells left some spaces among the cellular endfeet, and basement materials had accumulated in the spaces ( Figure 3 , Bottom left). Although the glial cells covered the damaged areas, there was no apparent ILM regeneration as a continuous flat sheet, with the exception of accumulated deposits of basement membrane materials around the Müller cell processes ( Figure 3 , Bottom left and Bottom right).
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