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 Table of Contents  
Year : 2019  |  Volume : 20  |  Issue : 3  |  Page : 95-99

Effect of subconjunctival bevacizumab injection before primary pterygium excision: a clinical and immunohistochemical study

1 Department of Ophthalmology, Faculty of Medicine, Al-Azhar University, Nasr City, Cairo Governorate, Egypt
2 Department of Ophthalmology, Faculty of Medicine, Al-Azhar University, Nasr City, Cairo Governorate
3 Department of Pathology, Faculty of Medicine, Al-Azhar University, Nasr City, Cairo Governorate, Egypt

Date of Submission20-Mar-2019
Date of Acceptance16-May-2019
Date of Web Publication26-Sep-2019

Correspondence Address:
MD Mona M Aly
Department of Ophthalmology, Faculty of Medicine, Al-Azhar University, Nasr City 11754, Cairo Governorate

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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/DJO.DJO_14_19

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Purpose The aim of this study was to evaluate the effect of preoperative subconjunctival bevacizumab injection on the clinical and immunohistochemical analysis of primary pterygium.
Patients and methods A total of 10 eyes of 10 patients with primary pterygium received a subconjunctival injection of bevacizumab (2.5 mg/0.1 ml) 1 week before simple pterygium excision with bare sclera technique. The control group (10 eyes of 10 patients with primary pterygium) had simple excision of primary pterygium with bare sclera without prior injection. Follow-up was performed on the first week, 2 weeks, 1 month, and 3 months postoperatively to evaluate the surgical site vascularization and occurrence of any complications and to record the rate of recurrence. Immunohistochemical analysis was performed on the excised tissue to detect vascular endothelial growth factor (VEGF) in both groups.
Results There was a marked decrease in pterygium vascularity after intralesional injection of bevacizumab. The injection group showed no recurrence after excision of pterygium during the scheduled follow-up period without any recorded complications regarding the use of bevacizumab, whereas the control group showed recurrence of pterygium in three eyes. The immunohistochemical analysis showed lower VEGF in the injected group than in the control group.
Conclusion Preoperative injection of bevacizumab effectively reduced vascularity and VEGF concentration of pterygium tissue. A subconjunctival preoperative single dose of bevacizumab was not associated with any adverse effects and was well tolerated. It had a minimizing effect on the extent of vascularization of the pterygium. No recurrence of pterygium was noted in any of the injected patients.

Keywords: bare sclera, bevacizumab, immunohistochemical analysis, pterygium, vascular endothelial growth factor

How to cite this article:
Tag Eldin AM, Aly MM, El Kholy MA. Effect of subconjunctival bevacizumab injection before primary pterygium excision: a clinical and immunohistochemical study. Delta J Ophthalmol 2019;20:95-9

How to cite this URL:
Tag Eldin AM, Aly MM, El Kholy MA. Effect of subconjunctival bevacizumab injection before primary pterygium excision: a clinical and immunohistochemical study. Delta J Ophthalmol [serial online] 2019 [cited 2020 Oct 25];20:95-9. Available from: http://www.djo.eg.net/text.asp?2019/20/3/95/267940

  Introduction Top

Pterygium is characterized by encroachment of a fleshy fibrovascular tissue from the bulbar conjunctiva onto the cornea. Although historically described as a degenerative disorder, it is more closely associated with inflammation and progressive fibrovascular proliferation [1]. Many growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor, platelet-derived growth factor, transforming growth factor β, and tumor necrosis factor α chemically stimulate angiogenesis and have been observed in fibroblastic and inflammatory pterygium cells [2]. Such growth factors were localized to epithelial cells, vessel endothelial cells, basement membrane of vessels, fibroblasts, and inflammatory cells of the pterygium [3]. However, the most prominent of these factors is VEGF, which is the main target of many current antiangiogenic therapies, including treatment with bevacizumab, a full-length humanized monoclonal antibody that binds VEGF and antagonizes its effects [4].

The definitive treatment of pterygium is achieved by surgical excision; however, surgery alone cannot prevent recurrence. To reduce the rate of recurrence, various modalities have been proposed [2].

This study was conducted to evaluate the effect of preoperative intralesional injection of bevacizumab as an adjuvant therapy for surgical excision on the clinical and immunohistochemical structure of primary pterygium.

  Patients and methods Top

This was a prospective, interventional, comparative study. An informed written consent was obtained from all patients before enrollment after explanation of the procedure, and the study was approved by the Local Ethics Committee of the Faculty of Medicine, Al-Azhar University.

The study included 20 eyes of 20 patients divided evenly into two groups, with 10 patients each. Group A included patients with pterygium excision preceded by a single preoperative subconjunctival bevacizumab (Avastin; Genentech Inc., San Francisco, California, USA) injection, whereas group B included patients with pterygium excision with no adjuvant therapy. In cases of bilateral pterygium, only the worst eye was treated.

All patients underwent complete ophthalmological examination before and after surgery, including visual acuity, slit-lamp examination of the anterior segment, posterior segment examination, and applanation tonometry.

Inclusion criteria were grade II or III primary pterygium (according to Tan et al. [5] grading of pterygium) with ocular irritation, cosmetic disfigurement or significant astigmatism.

Exclusion criteria were ocular surface pathology or infection, recurrent pterygium, autoimmune diseases, any condition for which bevacizumab was contraindicated, prior ocular trauma or surgery, and the inability to follow-up for the entire duration of the study.

Preoperative injection of bevacizumab

In the bevacizumab group, all the injections were performed in the operating room. Eyes were anesthetized with topical anesthetic eye drops. A subconjunctival injection of bevacizumab (Avastin 2.5 mg/0.1 ml) was administered intralesionally. Combined antibiotic and corticosteroid eye drops were prescribed four times daily for 1 week. All patients received a single preoperative injection only. Any complications regarding subconjunctival bevacizumab injection were recorded as well as the changes in the pterygium vascularity. One week after, the excision of pterygium was performed.

Surgical procedure

The pterygium excision was performed under topical and subconjunctival anesthesia to the area of pterygium. The bare sclera technique was used for excision of the pterygium in all patients. Postoperatively, patients were treated with topical combined antibiotic and corticosteroid eye drops four times daily for 1 week. Follow-up visits were at weeks 1 and 2, and months 1 and 3.

Immunohistochemical analysis

For histopathological examination of the excised tissue, 5-mm thick sections were prepared from each tissue paraffin block and stained with hematoxylin and eosin for confirmation of the diagnosis. For immunohistochemical staining, one section was cut from each case on positively charged slides and subjected to immunohistochemical staining using the streptavidin–biotin alkaline phosphate methods. The primary antibody used for immunohistochemical staining (with clone, manufacture, dilution, incubation period, and positive control) was rabbit antihuman VEGF polyclonal antibody, diluted 1 : 500 (Santa Cruz Biotechnology, Santa Cruz, California, USA). The sections were placed in an oven at 50°C for 30 min and then the sections were deparaffinized in xylene, rehydrated in graded alcohol dilution, washed in PBS, incubated with 0.3% hydrogen peroxide to block endogenous peroxidase activity, washed in PBS again, and boiled in citrate buffer solution (pH 6.0) using a microwave for 10 min at 60°C for antigen retrieval. After cooling at room temperature, the sections were incubated with primary antibody overnight in a humidified chamber and rinsed with PBS. The sections were then incubated for 30 min at 37°C with biotinylated secondary antibody and streptavidin conjugated to horseradish peroxidase. After three rinses with PBS, the sections were incubated with diaminobenzidine substrate and then rinsed with distilled water and counterstained with hematoxylin. The staining assessment of the three parameters was made on light microscope.

Interpretation of immunohistochemical staining

According to Aspiotis et al. [6], the pattern of expression was granular, with diffuse cytoplasmic disposition. Immunoreactivity for VEGF was presented in endothelial, stromal, and epithelial cells. To evaluate the expression of VEGF protein in epithelial cells, a combined score was established corresponding to the sum of both (a) staining intensity (0=negative, 1=weak, 2=intermediate, and 3=strong staining) and (b) percentile quadrants of positive cells (0=0%, 1=1–25%, 2=26–50%, and 3=>50%). The sum reached a maximum score of 6. Scores 1 and 2 were regarded as weak, 3 as intermediate, and 4–6 as strongly positive. For the estimation in stromal and endothelial cells, we used a semiquantitative score with a three-scale system: 0, no expression; 1, focal expression‏, and 2, diffuse expression. Patients’ data including the age, sex, grading of pterygium, postoperative clinical examination and the results of the immunohistochemical analysis were collected.

Statistical analysis

Data were collected, revised, coded, and entered into the statistical package for social science (IBM SPSS), version 20 (IBM Corp., Armonk, New York, USA). Qualitative data were presented as number and percentages. The comparison between two groups with qualitative data was done by using χ2-test. The confidence interval was set to 95% and the margin of error accepted was set to 5%. So, P value was considered significant at less than 0.05.

  Results Top

The data of all the included patients are demonstrated in [Table 1]. There was no statistically significant difference regarding age, sex, or preoperative grading of pterygium between both groups (P>0.05).
Table 1 The clinical findings of patients with primary pterygium

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There was a marked decrease in pterygium vascularity preoperatively following injection of bevacizumab, with no reported systemic or local complications; moreover, there was no reported necrosis, ischemia, or infection in the surgical bed area. There was no recorded intraoperative or immediate postoperative complications apart from subconjunctival hemorrhage in two eyes in each group. During the follow-up period, there was no recurrence in the bevacizumab group, whereas in the control group, there was a recurrence of pterygium in three (30%) cases.

The staining pattern of VEGF was diffuse, granular, and cytoplasmic, with intensification at the superficial layers of the epithelium. Intense immune-reactivity of epithelial, vascular endothelial, and many fibroblastic and inflammatory stromal cells was observed for VEGF ([Figure 1] and [Figure 2]). The mean percentage of VEGF-positive epithelial cells was 100%. Specifically, 9/10 (90%) cases presented high combined score (5–6) concerning VEGF ([Table 2]), in contrast to the relatively high frequency of intense staining stromal cells as well as vessels covered by VEGF-positive endothelial cells in pterygium tissue (80 and 90%, respectively; P=0.070 and 0.005, respectively, [Table 3]).
Figure 1 Immunohistochemical analysis of human pterygium for vascular endothelial growth factor. Vascular endothelial growth factor immunoreactivity appears to be positive in the endothelial cells of blood vessels, epithelial cells, and in the stromal inflammatory tissue (×100).

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Figure 2 (a) Vascular endothelial growth factor is moderately positive in epithelial cells, except for mucous cells. (b)Vascular endothelial growth factor is positive in the endothelial cells in blood vessels (×200).

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Table 2 Epithelial immune-reactivity of vascular endothelial growth factor in pterygium and post-treatment cases

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Table 3 Comparative results of expression levels of protein vascular endothelial growth factor in endothelial and stromal cells of pterygium and post-treatment cases

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In the epithelial part of pterygium after treatment, we have noted a weak reaction to VEGF with 50% of cases showing weak epithelial expression, whereas only one case gave high combined score (P=0.001, [Table 2]). In the vascular endothelial cells and subepithelial stromal cells, no such reaction has been observed, with 80 and 70% of cases giving negative expression for vascular endothelium and subepithelial stromal cells, respectively ([Figure 3]).
Figure 3 (a) Pterygium after treatment. (b) Vascular endothelial growth factor is weakly expressed in epithelium with decreased vascularity in the underlying stroma and marked fibrosis.

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  Discussion Top

The surgical treatment of pterygium focuses on the excision and prevention of recurrence [7]. To reduce the recurrence of pterygium, various adjunctive measures like mitomycin C, β-irradiation, and bevacizumab have been used [8]. The role of VEGF in pterygium pathogenesis has been evaluated, and elevated levels have been demonstrated in pterygium tissue as compared with normal conjunctivae [9].

In this study, the use of a single preoperative subconjunctival bevacizumab injection for primary pterygium was analyzed both clinically and immunohistochemically. The preoperative subconjunctival injection of bevacizumab was not associated with local or systemic adverse effects with no observed postoperative recurrence. Bevacizumab appeared well tolerated when administered subconjunctivally before pterygium excision. There was marked decrease in the pterygium vascularity after bevacizumab use.

Bevacizumab has been used at various doses, at variable times, and by different routes of administration. Ideal dose and treatment regimen should be important factors for the effects of bevacizumab on pterygium treatment [10]. This study demonstrated the effect of a single preoperative injection of bevacizumab in a dose of 2.5 mg/0.1 ml on pterygium size and vascularity, with marked decreases in both. Moreover, immunohistochemical analysis of the excised pterygium tissue with prior bevacizumab injection demonstrated decrease in VEGF expression in pterygium 1 week after injection.

In their study, Teng et al. [11] were the first to report a case of subconjunctival injection of bevacizumab in primary pterygium. A single injection of 1.25 mg/0.05 ml bevacizumab, subconjunctivally, into an inflamed primary pterygium at the limbus was used. This was well tolerated and reduced irritation and vascularization in the short term. The transient effect was likely related to the limited bioavailability of the drug in the setting of continued VEGF expression.

In this study, the single intralesional injection was followed by surgical excision of the pterygium 1 week after injection. In their study, Nuzzi and Tridico [2] concluded that the application of subconjunctival bevacizumab injections, at the dosage of 2.5 mg/0.1 ml, before and after surgical pterygium excision may be useful in preventing lesion recurrence after bare sclera procedure. Furthermore, bevacizumab subconjunctival administration may represent a safer alternative when compared with other surgical techniques and adjunctive drugs. In contrast to this study, a randomized clinical trial with the use of bevacizumab in a total dose of 7.5 mg subconjunctivally reported that bevacizumab had no significant effect on the recurrence rate of pterygium [12].

The limitations of this study included the small number of patients and the short follow-up period that did not allow for a definitive conclusion regarding the recurrence rate of pterygium with the use of preoperative subconjunctival bevacizumab.

  Conclusion Top

Preoperative injection of bevacizumab effectively reduced vascularity and VEGF concentration of pterygium tissue. Subconjunctival bevacizumab at a single preoperative dose was not associated with any adverse effects and was well tolerated. It had a minimizing effect on the extent of vascularization of pterygium. No recurrence of pterygium was noted in any of the injected patients.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Stival LR, Lago AM, Figueiredo MN, Bittar RH, Machado ML, Nassaralla Junior JJ. Efficacy and safety of subconjunctival bevacizumab for recurrent pterygium. Arq Bras Oftalmol 2014; 77:4–7.  Back to cited text no. 1
Nuzzi R, Tridico F. Efficacy of subconjunctival bevacizumab injections before and after surgical excision in preventing pterygium recurrence. J Ophthalmol 2017; 2017:1–7.  Back to cited text no. 2
Dos Santos Martins TG, de Azevedo Costa ALF, Furuzawa KM, Chammas R, Alves MR. Evaluation of antimitotic and antiangiogenic effect of preoperative subconjunctival application of mitomycin C in primary pterygium: a randomized trial. Int Ophthalmol 2019; doi: 10.1007/s10792-019-01081-0.  Back to cited text no. 3
Ozsutcu M, Ayintap E, Akkan JC, Koytak A, Aras C. Repeated bevacizumab injections versus mitomycin C in rotational conjunctival flap for prevention of pterygium recurrence. Indian J Ophthalmol 2014; 62:407–411.  Back to cited text no. 4
[PUBMED]  [Full text]  
Tan DT, Chee SP, Dear KB, Lim AS. Effect of pterygium morphology on pterygium recurrence in a controlled trial comparing conjunctival autografting with bare sclera excision. Arch Ophthalmol 1997; 115:1235–1240.  Back to cited text no. 5
Aspiotis M, Tsanou E, Gorezis S, Ioachim E, Skyrlas A, Stefaniotou M et al. Angiogenesis in pterygium: study of microvessel density, vascular endothelial growth factor, and thrombospondin-1. Eye (Lond) 2007; 21:1095–1101.  Back to cited text no. 6
Alsmman AH, Radwan G, Abozaid MA, Mohammed UA, Abd Elhaleim NG. Preoperative subconjunctival combined injection of bevacizumab and mitomycin C before the surgical excision of primary pterygium: clinical and histological results. Clin Ophthalmol 2017; 11:493–501.  Back to cited text no. 7
Sarac O, Demirel S, Oltulu R. Efficacy of intralesional bevacizumab administration in primary pterygium: a quantitative analysis. Eye Contact Lens 2014; 40:46–50.  Back to cited text no. 8
Galor A, Yoo SH, Piccoli FV, Schmitt AJ, Chang V, Perez VL. Phase I study of subconjunctival ranibizumab in patients with primary pterygium undergoing pterygium surgery. Am J Ophthalmol 2010; 149:926–931.  Back to cited text no. 9
Nava-Castañeda A, Ulloa-Orozco I, Garnica-Hayashi L, Hernandez-Orgaz J, Jimenez-Martinez MC, Garfias Y. Triple subconjunctival bevacizumab injection for early corneal recurrent pterygium: one-year follow-up. J Ocul Pharmacol Ther 2015; 31:106–113.  Back to cited text no. 10
Teng CC, Patel NN, Jacobson L. Effect of subconjunctival bevacizumab on primary pterygium. Cornea 2009; 28:468–470.  Back to cited text no. 11
Razeghinejad MR, Banifatemi M. Subconjunctival bevacizumab for primary pterygium excision; a randomized clinical trial. J Ophthalmic Vis Res 2014; 9:22–30.  Back to cited text no. 12


  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]


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