Corresponding Author: Hee Young Choi, MD, PhD. Department of Ophthalmology, Pusan National University School of Medicine, #179 Gudeok-ro, Seo-gu, Busan 602-739, Korea. Bevacizumab (Avastin; Genentech Inc., San Francisco, CA, USA) is a complete, humanized monoclonal antibody directed against all isoforms of vascular endothelial growth factor (VEGF). Bevacizumab was originally developed as a treatment for metastatic colorectal cancer [1], and it has been successfully applied off-label for intravitreal treatment of VEGF-mediated ocular diseases such as choroidal neovascular disorder, central retinal vein occlusion, proliferative diabetic retinopathy, and pseudophakic cystoid macular edema [2]. Recently, bevacizumab has been introduced as a new therapeutic strategy for many other ocular disorders, such as recurrent pterygium, neovascularization caused by chemical burns, viral infections of the cornea and bleb survival after glaucoma surgery [3-7]. Therefore, bevacizumab can be used to treat numerous ocular disorders, and it has been applied by various administration routes: anterior chamber injection, topical application or subconjunctival bevacizumab injection [3-7].
However, there have been many concerns regarding potential ocular side effects of bevacizumab. Several drugs that have been applied in ophthalmic treatments have demonstrated extraocular muscle toxicity. Anesthetics such as levobupivacaine and bupivacaine induce morphopathological changes in extraocular muscle [8,9]. However, subconjunctival injection of commercially available gentamicin and amphotericin causes acute toxic reactions in the extraocular muscles [10,11].
Despite this finding, other reports have demonstrated the safety profile of bevacizumab on the retina and cornea [12-17]; no data are currently available regarding the safety profile of bevacizumab on extraocular muscles. Recently, paralytic strabismus has been reported after bevacizumab injection in ocular disorders [18,19]. Therefore, this study was designed to evaluate the histological muscle safety of bevacizumab when injected directly into the extraocular muscle and subsequently diffused subconjunctivally in a rabbit model.
Materials and Methods
Thirty adult New Zealand white rabbits (2.0 to 3.0 kg, 20 weeks old) were used for this study. All rabbits were confirmed to be free from ocular disease. The animals were handled in accordance with the Association for Research and Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Surgical procedures
All surgical procedures were performed identically in both experimental and control eyes by a single surgeon (JHJ) as follows. In the first group (15 rabbits), the animals were treated with intramuscular injections of bevacizumab (1.25 mg/0.05 mL) in the right superior rectus muscle and with normal saline (0.05 mL) in the left superior rectus muscle. In the second group (15 rabbits), animals received subconjunctival injections of bevacizumab (2.5 mg/0.1 mL) in the superior conjunctiva of the right eye and normal saline (0.1 mL) subconjunctival injections in the left eye. Each rabbit was anesthetized with ketamine (400 mg/kg; Huons, Seoul, Korea) and xylazine (5 mg/kg; Bayer, Seoul, Korea) and placed in a stereotactic frame. The eyelids were opened using a speculum, and proparacaine was dropped into the conjunctival cul-de-sac. In the first group, the superior conjunctiva and Tenon's capsule were opened, and the superior rectus muscle was isolated from other tissues using cotton swabs and a muscle hook. Intramuscular injections were performed under direct visualization of the superior rectus muscle. After exposure, the superior rectus muscle was grasped with forceps, and a 30-gauge needle was placed 5 mm behind the insertion of the superior rectus muscle along the direction of the muscle fiber, and bevacizumab or normal saline was injected slowly. In the second group, subconjunctival injections were performed 3 mm behind the superior corneal limbus. The needle was removed 30 seconds after completion of injection to allow diffusion of the bevacizumab into the subconjunctival space and muscle and also to reduce leakage in both groups. After surgery, 3 mg/mL of ofloxacin (Tarivid; Santen, Osaka, Japan) eye drops were administered in each eye in both groups. All animals were observed and examined regularly for evidence of gross inflammatory reactions and scar formation by an investigator (HYC) in a blinded fashion. The animals were randomly sacrificed with an overdose of barbiturate anesthesia either one day, two weeks or four weeks post operation, and all eyes were enucleated and examined.
Light microscope examination
After sacrifice, the sites of muscle injection (5 mm behind the muscle insertion) and subconjunctival injection (3 mm behind the muscle insertion) were processed for light microscopic histopathologic evaluation of acute inflammatory reactions and late fibrotic scar formation of muscle tissue.
One day postoperatively, acute inflammatory reactions were evaluated in five rabbits from each group. Sections were stained with hematoxylin and eosin (H&E) for general histologic observation and also with antibodies for CD 11b that were specific for inflammatory cells: macrophages, monocytes and neutrophils. We performed H&E staining to assess global histologic changes of both groups two weeks after surgery.
Both eyes from every rabbit were evaluated for post inflammatory fibrotic scar formation and global histologic changes of each group four weeks postoperatively. Isolated tissues were examined using Masson's trichrome staining for scar tissue formation and H&E staining for general histologic observation. The fibrosis was graded based on the amount of collagen formation as follows: 0 = no fibrosis; 1 = mild perimuscular fibrotic reaction (stained collagen was detectable only in thin bands immediately adjacent to the muscle); 2 = easily detected thick bands; 3 = well-developed, dense bands of collagen; and 4 = a severe fibrotic response replacing large areas [20].
Electron microscope examination
We investigated ultra-structural muscle tissue changes after bevacizumab muscle injection (one day and four weeks after surgery) by electron microscope. The specimens were pre-fixed with 2.5% glutaraldehyde (4℃, phosphate buffer, pH 7.2) and were post-fixed with 1% osmium tetroxide in the same buffer. The materials were dehydrated with a graded ethyl alcohol series and embedded in epoxy resin (Epon 812 mixture). Thick sections (1 µm) were stained with 1% toluidine blue for light microscope examination. Thin sections (50 to 60 nm) were prepared by an ultramicrotome (Reichert SuperNova; Leica, Wein, Austria) and were double stained with uranyl acetate and lead citrate. Thin sections were also examined with a transmission electron microscope (JEM 1200EX-II; JEOL, Tokyo, Japan).
Results
The rabbits were monitored daily for changes within the orbit. All rabbits appeared to be healthy and ate normally. Mild conjunctival redness developed at the surgical site in some of the eyes in the first group. However, no significant ocular, periocular or orbital changes were noted in the treated eyes, and no gross changes were visible in the treated muscles at necropsy. There was no evidence of systemic toxicity in any animal.
Light microscopic examination
In the first group (muscle injection group), the extraocular muscle layers in all eyes were intact with no evidence of tissue injury or muscle fiber disorganization. Only minor edema occurred in sites that received direct needle penetration. The muscle fibers lacked hyper-contracted muscle fiber, showed even staining and were of uniform size in diameter with arrangement. However, there was minimal influx of CD-11 positive cells into the connective tissue of the extraocular muscle after direct muscle injections of bevacizumab and normal saline one day after the procedure. These cellular responses were of the same degree without significant differences between eyes that received bevacizumab injection and normal saline injection (Fig. 1).
Two weeks after the injections, we did not observe tissue necrosis or inflammatory cell infiltration in eyes that received bevacizumab and normal saline muscle injections; there were no remarkable differences between experimental and control eyes.
Masson trichrome staining revealed that the median value of fibrosis grading in the bevacizumab injection group and the normal saline injection group was 0 (no fibrosis), and the capillary networks and muscle global appearance of each muscle section were intact four weeks after bevacizumab injection and normal saline injection (Fig. 2).
In the second group (subconjunctival injection group), we observed no inflammatory cellular infiltration in H&E staining and no significant influx of CD-11 positive cells into muscle or connective tissue in eyes that received bevacizumab and normal saline injections one day after the procedure. There was no evidence of muscle necrosis and fibrosis in any of the sections from eyes that received bevacizumab and normal saline subconjunctival injections at two and four weeks after the procedure (Fig. 3).
Electron microscopic examination
Electron microscopic findings revealed normal muscles in all eyes. The nuclei and cell membranes remained morphologically unaffected, and the distinct banding patterns of sarcomeres were intact after the injections. Myofibrils and tubular systems were maintained, and mitochondria seemed to be preserved. In addition, the appearance of myonuclei was within the normal range, and we observed no significant differences between the control groups. The vascular beds, nerve endings and satellite cells appeared intact, and no significant differences were noted at all times in all sections (Fig. 4).
Discussion
The histological characteristics of mammalian extraocular muscles, which perform the functions of both red and white muscles, differ in many respects from those of other striated muscles. Extraocular muscle is distinguished by the finest fibers of any striated muscles, and the nerve supply to the extraocular muscles is astonishingly rich [21]. Extraocular muscles vary in diameter from 9 to 17 µm [22] and contain different types of muscle fibrils with intricate, ultramicroscopic structures and fibers with highly differentiated nerve endings [22,23]. Physiologically, extraocular muscle requires and receives more oxygen than other skeletal muscles and contracts much more quickly than other voluntary muscles [24]. All of these characteristics of extraocular muscles are related to fine extraocular muscle regulation, and any muscle injuries caused by extrinsic agents can lead to extraocular muscle dysfunction.
Some drugs that have been used in the ophthalmic arena have produced extraocular muscle toxicity [8-11]. Although the molecular patho-mechanisms of each drug are different, they follow similar histopathological courses. In acute stages, inflammatory cell infiltration is observed in muscle tissue, and necrobiotic changes may follow, ranging from fiber vacuolation and myocyte edema up to total disintegration of intracellular structures and myonecrosis [25,26]. Subsequently, satellite cells become activated, and muscle fibers are regenerated; in the final stages of inflammation, injured muscle demonstrates muscle atrophy and surrounding tissue fibrosis [27,28].
The present study found no significant tissue destruction on H&E staining or tissue fibrosis on Masson's trichrome stain after intramuscular and subconjunctival bevacizumab injection. CD-11b immunohistochemistry revealed no significant inflammatory cell infiltration in extraocular muscle tissue compared to control eyes. Although the intramuscular bevacizumab and normal saline injection groups showed minimal inflammatory cell infiltration in the connective tissue during the acute phase, this infiltration spontaneously disappeared and was probably related to direct needle injury or to the surgical procedure itself. Electron microscopic findings also revealed no significant necrosis of the muscle fiber and no remarkable vascular changes.
There are many ocular disorders in which VEGF plays a major role, such as retinal and choroidal vasoproliferative disorders, neovascular glaucoma and corneal neovascularization. Furthermore, VEGF is a vital factor in the inflammatory process and wound healing response [29,30], and it is present in extraocular muscle and retroorbital tissue [31]. Therefore, we expect that bevacizumab will be a useful future treatment modality in intraocular or extraocular ocular disorders.
In the current study, we observed no evidence of muscle injury or vascular network damage in any of the sections examined. Nevertheless, a normal histological appearance does not rule out functional changes to extraocular muscles. The effects of repeated exposure and the half-life of bevacizumab in the extraocular muscle are unknown; therefore, further studies will be required to evaluate the long-term safety of this drug and the possibility of functional side effects. In summary, we believe that the conventional dose of bevacizumab induced no extraocular muscle toxicity and was a safe agent for extraocular muscle.
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