Inflammatory responses after vitrectomy with vitreous substitutes in a rabbit model

In this study, 60 at least 4-month-old, pigmented rabbits obtained from a local breeder were used. The breeder is continually certified and controlled by the applicable governmental authorities, has many years of experience, and is a longtime supplier to our institution. All proceedings and animal treatment were in accordance with the guidelines and requirements of the government committee on animal experimentation at Lund University, and with the ARVO (The Association for Research in Vision and Ophthalmology) statement on the use of animals in ophthalmic and vision research.

Healaflow® (Aptissen S.A., Plan Les Ouates, Switzerland) is a transparent hydrogel, developed and marketed for use in glaucoma filtering surgery, where its purpose is to maintain the bleb and limit postoperative fibrosis [32‐34]. In this application, the gel is retained for 3–6 months [33], whereas it was retained in the vitreous space for at least a few weeks when evaluated as an experimental vitreous substitute [26]. The hydrogel consists of over 97% water, sodium hyaluronic acid (22.5 mg/ml) of non-animal origin cross-linked with BDDE (1,4-Butanediol diglycidyl ether), and phosphate and NaCl salts to maintain physiological pH (7.0) and osmolarity (305 mOsm/kg). Estimated specific gravity is circa 1.03, and refractive index i = 1.341.

Bio-Alcamid® (Polymekon, Brindisi, Italy) is a clear hydrogel, formerly used in plastic and reconstructive surgery as a tissue filler, mainly for lipoatrophic and posttraumatic conditions. The hydrogel consists of approximatively 4% reticulated polyalkylimide and approximately 96% non-pyrogenic water (pH 6.9), and is considered to contain no free monomers, and to be physically and chemically stable [35]. A collagen capsule surrounding the implanted Bio-Alcamid® is formed in vivo [36]. The usage decreased following the publication of several reports that described late complications after clinical use [37‐39], and the product is not currently available commercially. The stock used in this study was obtained previously.

Silicone oil, 1000 cSt (FCI S.A.S., Paris, France), is a well-established tamponade for vitreoretinal surgery. It is especially suitable when a long-term tamponade is indicated, such as complicated cases of retinal detachment and severe diabetic retinopathy [40, 41].

Two experienced surgeons (HB and SC) performed all surgical procedures. General anesthesia was used with a combination of ketamine (35 mg/kg) and xylazine (5 mg/kg) intramuscularly. Surgery was performed on the right eyes of the rabbits, while the left eyes served as controls. The pupils of the eyes were dilated with cyclopentolate (1%) and phenylephrine (10%) 30 min prior to surgery. Topical anesthesia with tetracaine (0.5%) was applied just before surgery. Sclerotomies for the infusion and the illumination probe were made with two transconjunctival 25 G trocars 1 mm posterior to the limbus (at 2 and 12 o’clock). For the main instrument, a sclerotomy was created with either a transconjunctival 25 G trocars, or a conjunctival cut and subsequent 19 G scleral incision (at 10 o’clock).

A BIOM 90-D lens (Oculus, Wetzlar, Germany) along with a standard endoilluminating probe were used for visualization. The Accurus Surgical System (Alcon, Fort Worth, TX, USA) with a vitreous cutter (Accurus 2500, Alcon, Fort Worth, TX, USA) was used, together with a continuous infusion of balanced salt solution (BSS plus, Alcon, Fort Worth, TX, USA). Core vitrectomy with posterior vitreous detachment (PVD) was performed, leaving peripheral parts of the vitreous intact due to the increased risk of traumatic cataract with the comparatively large lens of the rabbit. After a fluid-air exchange, approximately 0.5–1.0 ml of BSS (n = 14), Healaflow® (n = 12), or 1000 cSt silicone oil (n = 12) was injected into the vitreous cavity through a 25 G needle. Bio-Alcamid® (n = 8) was not possible to inject through the 25 G system due to its high viscosity. To accommodate for this, a modified cannula positioned through a 19 G sclerotomy was used, and the sclera and conjunctiva were closed with sutures at the end of the surgery. Limited touching of the crystalline lens (n = 8), and minor retinal touches (n = 8) occurred in some of the eyes slated for treatment for treatment with Healaflow®, silicone oil, and Bio-Alcamid® (Table 1). In addition, 4 eyes with limited lens touches were kept in the BSS group, while 14 eyes with significant perioperative complications (inadvertent displacement of the infusion into the subretinal space with subsequent retinal detachment (RD) (n = 2), significant retinal break (n = 13), and significant lens touch (n = 1)) were filled with BSS and analyzed separately (Table 1). Chloramphenicol ointment (Chloromycetin, Pfizer Inc., NY, USA) was applied immediately after surgery. No other postoperative treatment was given.

Clinical evaluation with ophthalmoscopy and intraocular pressure (IOP) measurement (Tono-Pen®) was performed postoperatively after 1 day, 1 week, and 1 month. At these time points, 4–6 rabbits in each group were euthanized. Due to the lack of a sufficient number of Bio-Alcamid®-filled eyes, no rabbits in this group were euthanized at the 1-day time point. At termination, the eyes were enucleated, examined macroscopically, photographed, dissected, and prepared for histological examination with routine microscopy and immunohistochemistry. In addition to the operated eyes, a total of 24 unoperated fellow eyes were processed for histology, with at least one sample from each group at each time point. These samples, together with at least one sample obtained from eyes of animals not subjected to surgery for each immunohistochemical marker, were used as controls.

The eyes were dissected vertically between the ora serrata regions including the optic nerve head and visual steak, and fixed for 4 h with 4% paraformaldehyde pH 7.3 in a 0.1 M Sørensen’s phosphate buffer (PB), and then repeatedly washed with 0.1 M Sørensen’s PB using the same solution containing sucrose of rising concentrations (5–25%). The specimens were sectioned at 12 μ on a cryostat, and every 10th slide was stained with hematoxylin and eosin according to standard procedures.

For immunohistochemical labeling, sections were washed in room temperature with 0.1 M of sodium phosphate-buffered saline pH 7.2 (PBS) with 0.1% Triton X-100 (PBS/Triton), and incubated overnight at + 4 °C with antibodies against the following antigens: CD45 (pan-leukocyte) double-labeled with galectin-3 (mainly microglia), CD68 (macrophages), CD20 (B lymphocytes), and GFAP (activated Müller cells), diluted with PBS/Triton with 1% bovine serum albumin (Table 2). After incubation with the primary antibodies, the specimens were rinsed with PBS/Triton, and the slides were thereafter incubated with fluorescein isothiocyanate (FITC)-conjugated antibodies (Sigma-Aldrich, St. Louis, MO, USA) for 45 min, rinsed, and mounted in anti-fading mounting media (VECTASHIELD, Vector Laboratories, Inc., Burlingame, CA, USA). For the double-labeling, the same procedure was performed with both of the primary antibodies, and in the latter step, both of the secondary antibodies (FITC-, and Texas Red-conjugated) added at the same time.

Negative controls were obtained by performing the same procedure as above but without any primary antibodies. A grading system was used for immunohistochemical analysis with the following designation for the estimated levels: (+) sporadic, + low, ++ moderate, +++ high, and ++++ very high (Table 3).

Eyes filled with BSS, as well as those treated with silicone oil, and eyes treated with Healaflow®, exhibited slight to moderate conjunctival swelling and occasional subconjunctival hemorrhage on the first day after surgery. The corneas were clear, and there were no overt signs of inflammation in the anterior chambers or the vitreous upon inspection macroscopically, and with binocular ophthalmoscopy. Mild vitreous hemorrhage was present in four cases. At later time points, there were no visible signs of superficial or intraocular inflammation, except in one eye treated with BSS which sustained severe cataract, uveitis, and possible endophthalmitis after 1 week. This animal was euthanized and excluded from further analysis. All of the eyes treated with Bio-Alcamid® exhibited signs of macroscopic inflammation such as conjunctival edema and injection. Additionally, all of these eyes showed cloudiness or suspect infiltrates in the vitreous. After a month, one of these eyes sustained massive inflammation with severe injection, conjunctival swelling, and corneal decompensation. The group of 13 eyes with significant perioperative retinal complications was macroscopically comparable to the BSS and Healaflow® groups.

Cataract formation was common especially in eyes with known perioperative touching of the lens, in some cases, hampering the view to the vitreous and retina. Two out of four eyes treated with BSS and Healaflow®, respectively, and all four treated with silicone oil remained clear after a month. All eyes filled with Bio-Alcamid® displayed cataract from 1 week and onwards. In the group that sustained significant perioperative retinal complications, a higher number of eyes had intraoperative touching of the lens and subsequent severe cataract formation. At 1 month, only one of these eyes remained completely clear.

Hematoxylin- and eosin-stained sections in unoperated in vivo controls displayed the expected normal morphology with preserved structure and lamination throughout the retina, and no pyknosis (Fig. 1a). A similar appearance was observed in eyes treated with BSS, Healaflow®, and silicone oil at all time points (Table 3, Fig. 2). In many of these eyes, especially the ones treated with silicone oil, clusters of cells were seen on the inner surface of the retina (Fig. 2a–i). Bio-Alcamid®-treated eyes exhibited a severely affected morphology with regions of profound thinning, disturbed lamination, and degeneration already at 1 week after surgery, progressing at the 1-month time point (Fig. 2j–k). These eyes also revealed a higher amount of cells on the inner retinal surface. In the 13 eyes with perioperative retinal complications, corresponding lesions such as retinal ruptures and RDs were seen with concurrent disturbances in the retinal lamination and severe degeneration in the affected areas (Fig. 2l–n).

Very low to no GFAP labeling was seen in unoperated control eyes (Fig. 1b), whereas labeling was present in almost all eyes that had gone through surgery (Table 3, Fig. 3). BSS-filled eyes, as well as those treated with Healaflow®, exhibited very low to low GFAP labeling in the inner retinal layers 1 day after surgery. After 1 week, moderately increased labeling was visible throughout the inner retina towards the outer plexiform layer (OPL). Heterogeneous minimal labeling remained in these layers after 1 month (Fig. 3a–f). Eyes treated with silicone oil displayed a similar labeling pattern at 1 day and 1 month, but with higher labeling at 1 week compared to those treated with BSS or Healaflow® (Fig. 3g–i). In eyes filled with Bio-Alcamid®, GFAP labeling revealed very high expression both at 1 week and 1 month postoperatively (Fig. 3j–k). The BSS-treated eyes with significant perioperative retinal complications showed a higher rate of labeling at all time points compared to eyes filled with BSS or Healaflow®, increasing over time with sustained high labeling present from 1 week to 1 month (Fig. 3l–n).

Galectin-3 labeling was not visible in unoperated control eyes (Fig. 1c). All operated eyes exhibited, in contrast to this, general diffuse labeling, especially in the inner retinal layers with no apparent difference between groups (Table 3, Fig. 4).

CD45 labeling was present in all eyes, with and without surgery (Table 3, Fig. 4), whereas only weak CD45 labeling of dendritic cells was observed in the inner retina (ganglion cell layer [GCL] and inner plexiform layer [IPL]) of unoperated eyes (Fig. 1c). In contrast to eyes from animals not subjected to surgery, all unoperated left eyes from the different study groups exhibited CD45-labeled cells in the inner retina and occasional cells on the inner limiting membrane (ILM) (Fig. 1f). All operated eyes displayed increased labeling in the inner retina, frequently with additional CD45 labeling of round cells on the ILM, and in the vitreous space (Fig. 4). Eyes treated with BSS, as well as those treated with Healaflow® exhibited low to moderate CD45 labeling at all time points (Fig. 4a–f). Silicone oil–treated eyes displayed moderate to high CD45 positivity in the inner retina on day 1, especially on the ILM, with the most pronounced labeling close to the optic nerve head (Fig. 4g, right). The CD45 labeling was high at 1 week, and again moderate to low at 1 month (Fig. 4g–i). All eyes treated with Bio-Alcamid® treatment showed very high-CD45 labeling intensity at 1 week and 1 month. In these eyes, labeling revealed both dendritic and round cells often spread throughout the entire retina, and enlarged multi-nucleated giant cells in the vitreous (Fig. 4j–k). In eyes with significant perioperative complications, the CD45 labeling was heterogeneous with moderate levels of labeled cells in the inner retina and on the ILM (Fig. 4l–n). There was a considerable regional variation in the labeling with aggregation of labeled cells in the vicinity of retinal lesions, where the labeling occurred in these layers as well as in deeper parts of the retina (Fig. 4n, right). Additionally, there were some eyes with occasional clusters of cells on the ILM and a tendency to form giant cells.

CD68 positive cells were seen sparingly in most operated eyes but not in unoperated controls (Table 3, Figs. 1d and 5). Labeled cells were found mainly in the inner retina, and in the vitreous. No distinct differences were seen between the eyes treated with BSS, Healaflow®, or silicone oil. All the eyes treated with Bio-Alcamid® exhibited extensive CD68 labeling, mostly in the vitreous and in the areas of the retina with most pronounced thinning and structural degeneration (Fig. 5j–k). In most eyes with significant perioperative retinal complications such as retinal breaks and RDs, increased labeling was present in parts of the retinas, mainly in the vitreous space, and on the ILM (Fig. 5l–n). No definite differences were seen between the different time points.

CD20 labeling was not present in unoperated eyes (Fig. 1e). A few labeled cells were present in a minority of the eyes treated with BSS, Healaflow®, and silicone oil. The majority of these cells was found in the vitreous space, in some cases in the GCL, and they were almost exclusively found at the first postoperative day. In the eyes treated with Bio-Alcamid®, extensive labeling with CD20 was present after a week. Few distinctly labeled cells remained after a month, although intense autofluorescence was present in multi-nucleated giant cells, and in some of the most degenerated parts of the retinas (Fig. 6j–k). CD20+ cells were detected on the first postoperative day in two of the eyes with significant perioperative retinal complications, with moderate labeling in one eye with RD. After 1 week, low labeling remained in one eye with both a rupture and RD, but none in other cases. No labeling was observed in this group after 1 month (Fig. 6l–n).