thirty adult new zealand white rabbits ( 2.0 to 3.0 kg , 20 weeks old ) were used for this study .
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 .
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 .
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 .
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 m ) 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 ) .
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 .
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 .
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 m ) 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 ) .
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 .
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 .
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 .
, 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 .
, 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 .
the nuclei and cell membranes remained morphologically unaffected , and the distinct banding patterns of sarcomeres were intact after the injections .
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 .
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 .
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 .
, 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 .
, 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 .
the nuclei and cell membranes remained morphologically unaffected , and the distinct banding patterns of sarcomeres were intact after the injections .
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 .
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 .
extraocular muscles vary in diameter from 9 to 17 m   and contain different types of muscle fibrils with intricate , ultramicroscopic structures and fibers with highly differentiated nerve endings .
physiologically , extraocular muscle requires and receives more oxygen than other skeletal muscles and contracts much more quickly than other voluntary muscles .
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 .
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 .
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 , and it is present in extraocular muscle and retroorbital tissue .
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 .
 purposethe purpose of this study was to investigate the myotoxicity of bevacizumab on extraocular muscles in a rabbit model.methodsthirty new zealand white rabbits were used for this study . 
 the animals were evenly divided into two groups . in the first group , 
 15 rabbits were treated with intramuscular injections of bevacizumab ( 1.25 mg/0.05 ml ) in the right superior rectus muscle and normal saline solution ( 0.05 ml ) in the left superior rectus muscle . in the second group , 
 15 rabbits were treated with subconjunctival injections of bevacizumab ( 2.5 mg/0.1 ml ) in the right superior subconjunctival area and normal saline solution ( 0.1 ml ) in the left superior subconjunctival area . 
 five rabbits in each group were sacrificed at one day , two weeks and four weeks after the injections . 
 extraocular muscle samples were prepared for light microscopic ( lm ) and electron microscopic ( em ) examination . 
 degrees of acute inflammation were evaluated via cd-11b immunohistochemistry , and global muscle change was investigated using hematoxylin and eosin stains . 
 intensity of fibrosis was evaluated using masson trichrome stains , and ultrastructural changes were observed on em.resultswe observed no significant inflammatory cell infiltration , muscle necrosis or fibrotic change in treated and control eyes . 
 em findings revealed no significant damage to muscle or vascular tissue after bevacizumab injection.conclusionswe found no signs of extraocular muscle toxicity after lm and em intramuscular and subconjunctival bevacizumab injections in a rabbit model . 
Materials and Methods
Surgical procedures
Light microscope examination
Electron microscope examination
Results
Light microscopic examination
Electron microscopic examination
Discussion