About MEAJO | Editorial board | Search | Ahead of print | Current Issue | Archives | Instructions to authors | Online submission | Subscribe | Advertise | Contact | Login 
Middle East African Journal of Ophthalmology Middle East African Journal of Ophthalmology
Users Online: 1304   Home Print this page Email this page Small font sizeDefault font sizeIncrease font size


 
ORIGINAL ARTICLE
Year : 2008  |  Volume : 15  |  Issue : 2  |  Page : 67-72 Table of Contents     

Impact of intravitreal injection of Bevacizumab (Avastin) on rabbit's choroid and retina


1 Ophthalmology Department, Research Institute of Ophthalmology, Cairo, Egypt
2 Pathology Department, Research Institute of Ophthalmology, Cairo, Egypt

Date of Web Publication13-Jul-2009

Correspondence Address:
Mostafa M Salaheldin
Ophthalmology Department, Research Institute of Ophthalmology, Cairo
Egypt
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0974-9233.51995

Rights and Permissions
   Abstract 

Aim: The aim of the study was to evaluate the impact of intravitreal injection of bevacizumab (Avastin) on chorio-capillaris permeability as well as structure changes in the choroid and the retina of pigmented rabbits.
Material and Methods: The study included 10 pigmented rabbits (20 clinically free eyes) ranged in weight between 1.2 and 2 kg (mean 1.7 0.05). The rabbits were subjected to intravitreal injection of 5 mg, 0.1mg Avastin in the right eyes (10eyes), while the left eyes (10eyes) were injected with equal volumes of balanced salt solution. 1 week later, Clinical examination and fundus fluorescein angiography (FFA) were done. Histological examination was performed on specimens of retina & choroid of Avastin & BSS injected eyes of sacrificed rabbits using light microscopy (LM) & transmission electron microscopy (TEM). Results were recorded and compared.th
Results: Post injection clinical examination of the eyes showed no abnormality of cornea, lens, vitreous and fundus. FFA showed remarkable decrease in background chorio-capillaris fluorescence in 7 eyes (70 percent) injected with Avastin as compared with eyes injected with BSS. No change was observed in regards to retinal vasculature, or abnormal dye leak. LM examination : specimens from Avastin group were evaluated in comparison to control eyes Treated eyes exhibited the same microscopic appearance in most specimens (8/10, 80 percent). The chorio-capillaris layer showed elongated, stretched monolayer of capillaries with flat, elongated endothelial cell lining. The laminae showed closely packed RBCs arranged in a monolayer with ribbon like shape. The surrounding interstitial tissue showed stretched, elongated & compact collagen fibers. The RPE cells were tightly adherent to each other with prominent nuclei. The different retinal layers were in concomitance with the control specimens, however mild to moderate disruption of photoreceptor outer segments together with mild vacuolization in the ganglion cell layer were seen. TEM examination of both control and treated specimens confirmed the findings recorded by LM. The endothelial cell limning of the choriocapillaris exhibited reduced fenestrations in between the cells. TEM also highlighted the compact lamellae of collagen fibers. The RPE cells showed remarkable increase in the number of mitochondria and prominent endoplasmic reticulum. Variable sized melanosomes were also seen th
Conclusion: Though single intravitreal injection of Avastin does not cause appreciable histological changes in rabbit retina and choroid, yet, it imposes definite effect on choriocapillaris permeability as evidenced by FFA and ultra structural changes. Repeated intravitreal injections might alter the hemostasis of the chorio-capillaris RPE complex.

Keywords: Bevacizumab, intravitreal injection, chorio-capillaris, retinal toxicity, vascular endothelial growth factor


How to cite this article:
Karawya S, Said DG, Salaheldin MM, Zaky I. Impact of intravitreal injection of Bevacizumab (Avastin) on rabbit's choroid and retina. Middle East Afr J Ophthalmol 2008;15:67-72

How to cite this URL:
Karawya S, Said DG, Salaheldin MM, Zaky I. Impact of intravitreal injection of Bevacizumab (Avastin) on rabbit's choroid and retina. Middle East Afr J Ophthalmol [serial online] 2008 [cited 2019 Nov 21];15:67-72. Available from: http://www.meajo.org/text.asp?2008/15/2/67/51995

The choroid vessels between the retina and the sclera of the eye originate from mesodermal tissue surrounding the newly formed optic cup early in development. The choroid endothelial cells are derived from the periocular mesenchyme. It is thought that the choroid vessels are induced by the RPE during development. Vascular endothelial growth factor (VEGF) might be a critical mediator of these RPE functions. [1] In tissue culture, multiple retinal cell types make VEGF and increase its production when the microenvironment is hypoxic. [2],[3] Angiogenesis is a highly complex and coordinated process requiring the sequential activation of a series of receptors in endothelial and mural cells. The data supporting a causal role for VEGF in ocular neovascularization are extensive. [4],[5],[6],[7]

Among the leading causes of blindness are retina and choroid diseases manifesting abnormal vessel permeability and growth. The advent of anti VEGF treatments marks a major advancement in the treatment of these eye diseases. [8],[9]

Little is known of the role of VEGF in the maintenance of adult ocular vasculature. VEGF is produced by human differentiated RPE cells and may be involved in paracrine signaling between RPE and chorio-capillaris. [10] Indirect evidence suggests that VEGF may be trophic for the chorio-capillaris and required for the maintenance of the chorio-capillaris fenestrae. [8]

The aim of the present study was to evaluate the impact of intravitreal injection of bevacizumab (Avastin) on chorio-capillaris permeability as well as structure changes in the choroid and the retina of pigmented rabbits.


   Material and Methods Top


The study included 10 pigmented rabbits (20 clinically free eyes) ranged in weight between 1.2 and 2 kg (mean 1.7 0.05). The rabbits were subjected to intravitreal injection of 5 mg, 0.1mg Avastin in the right eyes (10 eyes), while the left eyes (10 eyes) were injected with equal volumes of balanced salt solution (BSS).

The animals were examined daily and animals showing anterior segment pathology or infection were excluded from the study. FFA was done 1 week after the Intravitreal injection followed by sacrificing the animals with subsequent histopathological study of choroid & retina.

Intravitreal Injection

The animals were hypnotized by intra-muscular injection of 1ml (50mg) ketamin. Benoxinate hydrochloride 1% local anesthetic eye drops were installed into the conjunctival sac.

Paracentesis was done to prevent reflux of the injectant. Thereafter, 0.1 ml of the solution (Avastin or BSS) was injected into the vitreous cavity, using 30 caliber needles on insulin syringe. Injection site was selected 1mm outside the limbus.

Fundus Fluorescein Angiography (FFA)

The pupil of each eye was dilated with tropicamide hydrochloride 1% eye drops. The animals were sedated by a single I.M injection of promazine hydrochloride (2.33 mg/kg). The procedure started by injecting 1 ml flourescein solution (10 %) into the ear penna venule. Sequential photography of the fundus was done as soon as the dye reached the retinal circulation, after activation of the exciter and barrier filters using a Topcon TRC 50 camera. The angiographs were studied primarily to assess degree of background choroid fluorescence as an indicator of chorio-capillaris permeability. Fluorescein angiographs were randomly arranged and assessed by the three contributing retina specialists individually.

Histopathology

The animals were sacrificed (using an over dose of IV phenobarbitone). The eyes were enucleated & immediately bisected and fixed overnight with 4% gluteraldhyde in phosphate buffer PH 7.3. Sections of the posterior segment (2x2) were taken 2mm inferior to the optic disc, and post fixed with 1.3% osmium tetroxide in phosphate buffer PH 7.3, for 3 hours, embedded in Epon after dehydration in a graded series of acetones. Later, semi-thin Sections were obtained, stained with Toluidine blue stain and examined by light microscopy (LM). Further ultrathin sections were cut and double stained with Urynit acetate and Lead citrate and examined with Transmission Electron Microscope (TEM).

LM and TEM examination aimed to evaluate primarily the chorio-capillaris layer, in addition to RPE cells, and the neurosensory retina.


   Results Top


Post injection clinical examination of the eyes showed no abnormality of cornea, lens, vitreous and fundus.

Concerning FFA, there was an agreement of remarkable decrease in background chorio-capillaris fluorescence in 7 eyes (70 percent) injected with Avastin as compared with eyes injected with BSS. No change was observed as regards retinal vasculature, or abnormal dye leak [Figure 1]a & b.

Light Microscopic Examination: specimens from Avastin group were evaluated in comparison to control eyes. Treated eyes exhibited the same microscopic appearance in most specimens (8/10, 80%). The chorio-capillaris layer showed elongated, stretched monolayer of capillaries with flat, elongated endothelial cell lining [Figure 2],[Figure 3]. The laminae showed closely packed RBCs arranged in a monolayer with ribbon like shape. The surrounding interstitial tissue showed stretched, elongated, compact collagen fibers [Figure 4],[Figure 5].

The RPE cells were tightly adherent to each other with prominent nuclei. The different retinal layers were in concomitance with the control specimens, however mild to moderate disruption of photoreceptor outer segments together with mild vacuolization in the ganglion cell layer were seen [Figure 5].

TEM examination of both control and treated specimens confirmed the findings recorded by LM, however further details were recorded [Figure 6],[Figure 7],[Figure 8],[Figure 9].

The endothelial cell lining of the chorio-capillaris exhibited reduced fenestrations in between the cells [Figure 8]. TEM also highlighted the compact lamellae of collagen fibers.

Concerning the RPE cells, there was remarkable increase in number of mitochondria and prominent endoplasmic reticulum. Variable sized melanosomes were also seen [Figure 9].


   Discussion Top


The histological structure of the rabbit choroid and retina is essentially the same as that of the human choroid. Thickness ranges from 48 above to 120 below the optic disc. The chorio-capillaris forms a single layer of fine capillaries. [11]

VEGF is a homo-dimeric glycoprotein. It is a critical regulator of vasculo-genesis and angiogenesis. [12],[13],[14] It is a potent inducer of vascular permeability via multiple mechanisms, including endothelial injury (15), fenestrae formation, [16] dissolution of tight junctions, and trans-cellular bulk flow. [17],[18]

In addition to vasculo-genesis and angiogenesis, VEGF may participate in the maintenance of certain vascular systems in the human adult. Specific binding of VEGF is associated with mature vessels in various adult rat tissues, suggesting that VEGF has a function in the maintenance of quiescent vascular endothelium. [19]

Interruption of VEGF production in the retina and RPE in developing animals has been shown to cause serious defects in retinal and choroid development. Elimination of VEGF in adult animals by genetic or pharmacologic means has not been shown to result in changes in retinal or choroid vasculature. [20],[21] However, Peters et al in a study of the effect of intravitreal bevacizumab on ultra structures of primate eye; recorded change in the chorio-capillaris in the from of reduction of chorio-capillaris endothelial cell fenestrations. [22]

In a normal eye the RPE secretes VEGF at its basal side, which is required for the maintenance of the chorio-capillaris. [10] Absence of VEGF causes secondary atrophy of the chorio-capillaris [23] and results in a loss of endothelial cell fenestrations. [24]

In the non-human primate eye, intravitreal injections of VEGF are capable of triggering a severe diabetic phenotype, including neovascularization of the retina and iris. [25],[26]

Therapeutic antagonism of VEGF in animal models results in significant inhibition of both retinal and choroid neovasculariztion as well as reduction in vascular permeability. [27],[28]

In our study, intravitreal injection of Avastin in rabbit eyes resulted in decreased choroid capillaries permeability as suggested by the remarkable decrease in background choroid fluorescence which might be secondary to decreased free passage of dye via chorio-capillaris fenestrae.

Histopathologically, no significant changes were encountered in the retina and choroid of treated eyes by light microscopy.

Several investigators suggested that a single intravitreal injection of bevacizumab at doses up to 5 mg in rabbit eyes dose not result in obvious retinal toxicity. The end point ERG did not have a significant decrease. However, the lack of changes by light microscopy does not exclude possible alterations on a submicroscopic level. [29],[30],[31]

On the other hand, in our study, TEM sections showed decreased chorio-capillaris permeability augmented by the blockage of fenestrae between chorio-capillaris endothelial cells, as well as compactness of collagen fibers in the stroma as compared to controls. This would be in accordance with the work done by Peters et al. [22]

Another TEM feature was the increase in intracellular content of mitochondria and prominent endoplasmic reticulum of RPE cells reflecting increased activity of the cells which might be a feed back mechanism induced by decreased VEGF concentration around RPE cells.


   Conclusion Top


Though single intravitreal injection of Avastin does not cause appreciable histological changes in rabbit retina and choroid, yet, it imposes definite effect on chorio-capillaris permeability as evidenced by FFA and ultra-structural changes. Repeated intravitreal injections might alter the hemeostasis of the chorio-capillaris RPE complex.

 
   References Top

1.Zhao S, Overbeek PA; Regulation of choroid development by the retinal pigment epithelium. Mol Vis 2001;7:277-282.  Back to cited text no. 1    
2.Shima D, Adamis AD, Ferrara N, et al. Hypoxic induction of endothelial cell growth factors in retinal cells: identification and characterization of vascular endothelial growth factor (VEGF) as the sole mitogen. Mol Med 1995;2:182-193.  Back to cited text no. 2    
3.Aiello LP, Nothrup JM, Keyt BA. Hypoxic regulation of vascular endothelial growth factor in retinal cells. Arch Ophthalmol 1995;113:1538-1544.  Back to cited text no. 3    
4.Ferrara N. VEGF and the quest for tumor angiogenesis factors. Nat Rev cancer 2002;2:795-803.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Leung DW, Cachianes G, Kuang WS, et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246:1306-1309.  Back to cited text no. 5    
6.Yancopoulos GD, Davis S, Gal NW, et al. Vascular specific growth factor and blood vessel formation. Nature 2000;407:242-248.  Back to cited text no. 6    
7.Ferrara N. vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 2004;25:581-611.  Back to cited text no. 7  [PUBMED]  [FULLTEXT]
8.Adamis A, Shima D. The role of vascular endothelial growth factor in ocular health and disease. Retina 2005;25:111-118.  Back to cited text no. 8    
9.Bhisitkul RB. Vascular endothelial growth factor biology: clinical implications for ocular treatments. Br J Ophthalmol 2006;90:1542-1547.  Back to cited text no. 9  [PUBMED]  [FULLTEXT]
10.Blaavwgeers HG, Holtkamp GM, Rutten H, et al. Polarized vascular endothelial growth factor secretion by human retina pigment epithelium and localization of vascular endothelial growth factor receptor on the inner choriocapillaris: evidence for a trophic paracrine relation. Am J Pathol 1999;155:421-428.  Back to cited text no. 10    
11.Hyvarinen I. Circulation in the fundus of the rabbit eye. Acta Ophthalmol 1967;45:862.  Back to cited text no. 11    
12.Sanger DR, Galli SJ, Dvorak AM, et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219:983-985.  Back to cited text no. 12    
13.Leung DW, Cachians G, Kuang WT, et al. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246:1306.  Back to cited text no. 13    
14.Ferrara N, Heuzel WJ. Pituitary follicular factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989;161:851-858.  Back to cited text no. 14    
15.Joussen AM, Murata T, Tsujikawa A, et al. Leukocyte mediated endothelial cell injury and death in the diabetic retina. Am J Pathol 2001;158:147-152.  Back to cited text no. 15  [PUBMED]  [FULLTEXT]
16.Roberts WG, Palade GE. Neovasculature induced by vascular endothelial growth factor is fenestrated. Cancer Res 1997;57:765-772.  Back to cited text no. 16  [PUBMED]  [FULLTEXT]
17.Autonetti DA. Barber AJ. Hollinger LA, et al. Vascular endothelial growth factor induces rapid phosporylation of tight junction proteins in zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors. J. Biol Chem 1999;274:23463-23467.  Back to cited text no. 17    
18.Qu H, Nagy JA, Senger DR, eat al. Ultra structural localization of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) to the luminal plasma membrane and vesiculovacular organelles of tumor microvascular endothelium. J Histochem Cytochem 1995;43:381-389.  Back to cited text no. 18    
19.Jakeman LB, Winer J, Bennett GL, et al. Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues. J Clin Invest 2000;106:1311-1319.  Back to cited text no. 19    
20.Haigh JJ, Morelli PI, Gerhardt H, et al. Related cortical and retinal defects caused by dosage-dependent reduction in VEGF - a paracrine signaling. Dev Biol 2003;262:225-241.  Back to cited text no. 20  [PUBMED]  [FULLTEXT]
21.Marreros AG, Fan J, Yokoyama Y, et al. Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function. Am J Pathol 2005;167:1451-1459.  Back to cited text no. 21    
22.Petrs S, Heiduscka P, Julien S, et al. Ultra structural findings in the primate eye after intravitreal injection of bevacizumab. Am J Ophthal 2007;143:995-1002.  Back to cited text no. 22    
23.Korte GE, Gerszberg T, Pua F, Henkind P. Choriocapillaris atrophy after experimental destruction of the retinal pigment epithelium in the rat: a study in thin sections and vascular casts. Acta Anat 1986;127:171-175.  Back to cited text no. 23  [PUBMED]  
24.Leonard DS, Zhany XG, Panozzo G, et al. Clinico-pathologic correlation of localized retinal pigment epithelium debridement. Invest Ophthalmol Vis Sci 1997;38:1094-1109.  Back to cited text no. 24    
25.Tolentino MJ, Miller JW, Gragoudas ES, et al. Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microagiopathy in an adult primate. Ophthalmology 1996;103:1820-1828.  Back to cited text no. 25  [PUBMED]  
26.Tokutino MJ, Miller JW, Gragoudas ES, et al. Vascular endothelial growth factor to produce iris neo-vascularization and neovascular glaucoma in a nonhuman primate. Arch Ophthalmol 1996;114:964-970.  Back to cited text no. 26    
27.Aiello LP, Pierce EA, Foley ED, et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor proteins. Proc Natl Acad Sci USA 1995;92:10457-10461.  Back to cited text no. 27  [PUBMED]  [FULLTEXT]
28.Qaum T, Xu Q, Joussen AM, et al. VEGF - initiated blood-retinal breakdown in early diabetes. Invest Ophthalmol Vis Sci 2001;42:2408-2413.  Back to cited text no. 28  [PUBMED]  [FULLTEXT]
29.Manzano RP, Peyman GA, Khan P, Kivilcim M. Testing intravitreal toxicity of bevacizumab (Avastin). Retina 2006;26:257-261.  Back to cited text no. 29  [PUBMED]  [FULLTEXT]
30.Bakri SJ, Cameron JD, Mccannek CA, et al. Absence of histologic retinal toxicity of intravitreal bevacizumab in a rabbit model. Am J Ophthalmol 2006;142:162-164.  Back to cited text no. 30    
31.Feiner L, Barr, E, shui Y, et al. safety of intravitreal injection of bevacizumab in rabbit eyes. Retina 2006;26:882-888.  Back to cited text no. 31    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]


This article has been cited by
1 SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHIC ANALYSIS OF HEALTHY RETINA IN BRANCH RETINAL VEIN OCCLUSION AND ITS RESPONSE TO ANTIANGIOGENIC THERAPY
Guillermo Salcedo-Villanueva,Mariana Harasawa,Raul Velez-Montoya,Marc T. Mathias,Frank S. Siringo,Jeffrey L. Olson,Scott C. Oliver,Naresh Mandava,Hugo Quiroz-Mercado
Retina. 2015; 35(4): 704
[Pubmed] | [DOI]
2 Histological findings of uveal capillaries in rabbit eyes after multiple intravitreal injections of bevacizumab
Yosuke Sugimoto,Hideki Mochizuki,Hidetaka Miyagi,Seiichi Kawamata,Yoshiaki Kiuchi
Current Eye Research. 2013; 38(4): 487
[Pubmed] | [DOI]
3 Effects of bevacizumab on neuronal viability of retinal ganglion cells in rats
Maria Rosaria Romano,Francesca Biagioni,Gianluca Besozzi,Albino Carrizzo,Carmine Vecchione,Francesco Fornai,Marcello Diego Lograno
Brain Research. 2012; 1478: 55
[Pubmed] | [DOI]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
    Material and Methods
    Results
    Discussion
    Conclusion
    References
    Article Figures

 Article Access Statistics
    Viewed2502    
    Printed163    
    Emailed0    
    PDF Downloaded186    
    Comments [Add]    
    Cited by others 3    

Recommend this journal