CLINICAL SCIENCE
Angioregressive Pretreatment of Mature Corneal Blood
Vessels Before Keratoplasty: Fine-Needle Vessel
Coagulation Combined With Anti-VEGFs
Yanyan Koenig, MD,* Felix Bock, MD,*† Friedrich E. Kruse, PhD,* Katja Stock, MD,‡
and Claus Cursiefen, MD*†
Purpose: To evaluate the efficacy and safety of combined feeder
vessel coagulation and topical antiangiogenic therapy using bevaci-
zumab in the treatment of mature corneal blood vessels.
Methods: Sixteen eyes of 16 patients with mature corneal neo-
vascularization (NV) due to different underlying corneal diseases
underwent fine-needle feeder vessel coagulation by diathermy and
were treated postoperatively for up to 4 weeks with topical bev-
acizumab eye drops (containing 5 mg/mL bevacizumab) 5 times
a day. Nine patients received an additional subconjunctival bev-
acizumab injection at the time of cautery.
Results: The mean duration of follow-up was 276 ± 147.3 days
(range, 29–464 days). Regression of the feeder vessel was observed
in 14 eyes. The vascularized area was reduced significantly (P ,
0.05). Combined subconjunctival and eye drop antivascular endo-
thelial growth factor treatment was significantly more effective in
reducing the vascularized area compared with antivascular endothe-
lial growth factor eye drop therapy alone (P , 0.05). Five patients
(5 eyes) needed a second treatment. Thirteen patients (13 eyes)
receiving topical bevacizumab treatment combined with feeder ves-
sel coagulation showed stable visual acuity. Two patients had
improved visual acuity. One patient had enlarged area of lipid kerat-
opathy despite successful treatment of corneal NV and thus
decreased visual acuity. Overall, there was a nonsignificant improve-
ment of best-corrected visual acuity (P . 0.05).
Conclusions: In this pilot study, fine-needle feeder vessel coagulation
combined with topical bevacizumab application for treatment of mature
corneal NV seemed to be a well-tolerated new treatment option to
regress corneal NV. This may not only improve corneal transparency
but also “preconditions” such a cornea for future keratoplasty.
Received for publication January 30, 2011; revision received June 8, 2011;
accepted July 8, 2011.
From the *Department of Ophthalmology, University of Erlangen-Nürnberg,
Schwabachanlage, Erlangen, Germany; †Department of Ophthalmology,
University of Cologne, Cologne, Germany; and ‡University Pharmacy,
University of Erlangen-Nürnberg, Erlangen, Germany.
Supported by IZKF Erlangen. The funding organization had no role in the
design or conduct of this research.
The authors declare no conflicts of interest.
Reprints: Yanyan Koenig, or Claus Cursiefen, Department of Ophthal-
mology, University of Erlangen-Nürnberg, Schwabachanlage 6, 91054
Erlangen, Germany (e-mail: yanyan.koenig@uk-erlangen.de or claus.
cursiefen@uk-koeln-de).
Copyright © 2012 by Lippincott Williams & Wilkins
Cornea  Volume 31, Number 8, August 2012
Key Words: corneal neovascularization, subconjunctival bevacizumab,
bevacizumab eye drop, feeder vessel coagulation
(Cornea 2012;31:887–892)
T he normal human cornea is avascular. This avascularity
is actively maintained by expression of antiangiogenic and
antilymphangiogenic factors.1,2 Corneal neovascularization
(NV) can occur after numerous corneal diseases and leads to
decreased visual acuity and poor prognosis in subsequent cor-
neal transplantation since corneal immune privilege is lost.3
Although steroids are the first choice against pro-
liferative corneal blood vessels in clinical routine, they are
not always effective and have serious long-term side
effects. Members of the vascular endothelial growth factor
(VEGF) family are thought to play a key role in pathological
NV.4,5 Bevacizumab (Avastin; Roche Pharma AG, Basel,
Switzerland) is a humanized monoclonal antibody blocking
VEGF-A, which results in inhibition of the outgrowth of
new vessels. Bevacizumab is approved for the treatment of
metastatic colorectal cancer. It is currently injected “off label”
into the vitreous humor for the treatment of proliferative and
nonproliferative diabetic retinopathy, neovascular age-related
macular degeneration, and neovascular glaucoma with prom-
ising results.2
We recently demonstrated that bevacizumab eye drops
significantly inhibit progressive corneal angiogenesis in a
series of 30 eyes. No obvious side effects were observed.6
There are increasing numbers of reports on the efficacy of
subconjunctival bevacizumab injections and eye drops in
animal models and in patients.
In contrast, limited treatment options exist for mature
corneal vessels, which—because of their pericyte-covered
vessel wall—do not depend on VEGF anymore.7,8 Here, we
report on the short- and long-term visual acuity changes and
changes in vascularized corneal area after feeder vessel
coagulation for the treatment of mature pathological corneal
NV combined with topical bevacizumab application. We hypo-
thesize that additional anti-VEGFs dampen the angiogenic
stimulus caused by the cautery itself. Because experimental
studies clearly show that thermal cautery of the cornea causes
influx of proangiogenic inflammatory cells and angiogenesis,3,9
we reasoned that a combined antiangiogenic treatment at the
time of cautery could reduce the risk of vessel reperfusion.
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Koenig et al
PATIENTS AND METHODS
The uncontrolled noncomparative case series consisted
of 16 eyes of 16 patients (10 men, 6 women) between 17 and
82 years of age (mean age, 48 ± 20 years) with established
corneal NV due to herpetic keratitis (n = 10), limbal stem cell
deficiency after chemical burn (n = 3), and corneal graft
rejection (n = 3). Established corneal NV was defined as stable
corneal vessels for at least 6 months despite conventional
treatment including steroids or systemic immunosuppression.
All the 16 patients underwent fine-needle feeder vessel
coagulation.8 The tip of a curved needle attached to a 10-0
nylon suture (Alcon, Fort Worth, TX) was inserted into the
lumen of the blood vessel. A unipolar diathermy probe was
brought into contact with the end of the needle (15 mW). The
contact was kept until the vessel lumen collapsed. The dia-
thermy machine was an ICC 50 with a 90 degree–angled
needle electrode (ERBE GmbH, Tübingen, Germany).
Nine patients received an additional subconjunctival
bevacizumab injection (2.5 mg/0.1 mL) during the same
surgical session. All the 16 patients were postoperatively
treated for up to 4 weeks (mean, 13.6 ± 8.5 days) with topical
bevacizumab eye drops (containing 5 mg/mL bevacizumab)
5 times a day, which were given in addition to current local
and systemic therapy. Patients with corneal NV due to her-
petic keratitis received local and systemic antiviral therapy in
addition. Treatment for patients with corneal NV due to chem-
ical burn or graft rejection consisted of local and systemic
steroids or other immunosuppressive agents. In case of visible
reperfusion of the occluded feeder vessel, a second treatment
was performed 4 weeks after the first treatment. Figure 1
shows a flow chart of our therapy regimen. Mean follow-up
time was 276 ± 147.3 days (range, 29–464 days). Bevacizu-
mab was used off label as subconjunctival injections and eye
drops. The institutional review board approved this treatment,
and all patients gave a written consent before treatment.
Bevacizumab eye drops were prepared in the hospital
pharmacy, diluting the standard solution in 0.9% saline to
a concentration of 5 mg/mL. Bevacizumab was portioned into
1-mL syringes for daily use. The eye drops were stored at
4°C. After opening, they were kept at 4°C and used within 2
weeks.6
At each visit, a routine Snellen visual acuity assessment
was performed followed by an ophthalmic examination, inclu-
ding fluorescein staining and tonometry. Visual improvement
was defined as halving of the minimum angle of resolution.
Worsening of visual acuity was defined as doubling of the visual
angle. Changes in corneal NV were determined by repeatedly
performing standardized digital slit-lamp pictures, which were
then analyzed morphometrically using an image analysis soft-
ware based on gray filter sampling (Cell^F; Olympus, Hamburg,
Germany) as described previously.10,11 The area of vasculariza-
tion was measured in terms of pixels. Its ratio to the entire
corneal area resulted in the percentage of corneal NV.
Statistical analysis was performed by Microsoft Excel
(Microsoft Corporation, Redmond, WA) and R (R Founda-
tion for Statistical Computing, Vienna, Austria). Graphs were
drawn using R (R Foundation for Statistical Computing).
Statistical significance was defined as P , 0.05.
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Cornea  Volume 31, Number 8, August 2012
RESULTS
Antiangiogenic Efficacy
The mean duration of follow-up was 276 ± 147.3 days
(range: 29–464 days), and the median follow-up time was
240 days. The mean treatment duration with bevacizumab
eye drops was 13.6 ± 8.5 days. Corneal photographs of all
16 eyes (16 patients) could be assessed by image analysis.
Fine-needle vessel coagulation combined with topical beva-
cizumab treatment led to regression of mature corneal vessels
in 14 of 16 patients. Five patients (5 eyes) needed a second
treatment. Still, 2 of these patients showed continuous pro-
gression of corneal NV. One patient suffered from herpetic
keratitis and 1 from limbal stem cell insufficiency after chem-
ical burn. The mean overall reduction in vascularized area
after treatment was 46% and was statistically significant
(P , 0.05; Fig. 2). The decrease of vascularized corneal area
was especially striking in the group of patients with herpetic
keratitis (Fig. 3). The results in the other 2 groups were con-
sidered not significant. Subconjunctival bevacizumab injec-
tion at the time of cautery together with eye drops led to
a significantly greater reduction in corneal NV compared with
cautery and anti-VEGF eye drops (P , 0.05; Fig. 4).
Occlusion of all treated vessels was achieved in 11
patients (68.8 %). During follow-up (of up to 15 months), there
was no reperfusion of the occluded feeder vessel. Figure 5
shows a patient with herpetic keratitis preoperatively and
15 months postoperatively with no detectable corneal vessels
anymore. None of the 16 patients complained of pain or
discomfort throughout the follow-up or about any drug-related
ocular or systemic adverse events. No allergic reactions were
observed.
Changes of Visual Acuity
In total, 2 eyes showed improved visual acuity (halving
of the minimum angle of resolution), 13 eyes showed stable or
slightly improved visual acuity, and 1 eye showed enlarged
area of lipid keratopathy (after 2 coagulations of the feeder
vessel) despite successful treatment of corneal vascularization,
and thus decreased visual acuity (doubling of the minimum
angle of resolution). Overall, there was a nonsignificant
improvement of visual acuity (P . 0.05; Fig. 6).
Most patients in our study (10 eyes) had corneal NV due
to herpetic keratitis. We intended to see whether there would
be any difference in visual outcome depending on the
underlying cause of corneal NV, but there was no significant
change in visual acuity in any of the 3 diagnostic groups.
Furthermore, we investigated whether there were any benefi-
cial effects of additional subconjunctival bevacizumab in
addition to anti-VEGF eye drops on best-corrected visual
acuity (BCVA). There was no significant change in BCVA
between the groups with and without additional subconjunc-
tival bevacizumab injection.
DISCUSSION
Various inflammatory, infectious, degenerative, or
traumatic disorders are associated with corneal NV. This
Ó 2012 Lippincott Williams & Wilkins
Cornea  Volume 31, Number 8, August 2012
FIGURE 1. Flow chart of the thera-
peutic regimen and respective number
of patients included in each treatment
category used in this study.
sight-threatening complication occurs when the complex
balance between angiogenic and antiangiogenic factors within
the cornea is tilted toward angiogenic molecules. The
concentrations of VEGF were shown to be significantly
increased in inflamed and vascularized human and animal
Ó 2012 Lippincott Williams & Wilkins
Angioregressive Pretreatment Prior to Keratoplasty
corneas.12,13 In the past, different substances have been tried
as angiogenesis inhibitors, including steroids, cyclosporin A,
and anti-VEGFs. In particular, the latter have recently been
shown to be effective primarily against proliferating corneal
blood vessels. Bevacizumab is a full-length humanized
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Koenig et al
FIGURE 2. Fine-needle vessel coagulation combined with
topical bevacizumab treatment leads to a significant reduction
of corneal NV. The diagram shows vascularized corneal area
pre- and postoperatively.
monoclonal antibody against VEGF-A, approved for intrave-
nous administration for the treatment of colorectal cancer.
Dastjerdi et al14 investigated the safety and efficacy of topical
bevacizumab in the treatment of corneal NV. In that prospec-
tive study, 10 patients received bevacizumab eye drops. There
were significant reductions of NV area and vessel caliber.
Yet, there were no significant changes in visual acuity.
Recently, we showed in 30 eyes that bevacizumab reduced
corneal NV when given as eye drops against proliferative
corneal NV.6 Bahar et al15 reported on the efficacy of subcon-
junctival bevacizumab injections (2.5 mg/0.1 mL) in
10 patients with corneal NV. Visual acuity did not change
significantly in any patient.
So far, there is no specific angioregressive treatment
available against mature corneal NV. Although steroids are
the first choice in clinical practice, they are not always
effective, and long-term use may cause serious side effects.
Alternative treatment options against mature corneal blood
vessels include photodynamic therapy, argon laser treatment,
and cautery.8,16,17 In the only other report on cautery of cor-
neal blood vessels, Pillai et al8 used fine-needle diathermy to
treat 14 patients with established corneal vessels due to dif-
ferent causes. Occlusion of all vessels was achieved in
8 patients. In 4 patients, 75% of the treated vessels were
occluded, and in the remaining 2 patients, 50% were
occluded. No significant change in BCVA was noticed.8
In our study, we chose to use a combination of cautery
and anti-VEGFs to dampen the additional angiogenic stimu-
lus caused by the cautery procedure itself. All patients
included in our study had stable corneal NV for at least
6 months. Previous treatment attempts including topical and
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Cornea  Volume 31, Number 8, August 2012
FIGURE 3. Decrease of the vascularized corneal area between
pre- and postoperative measurements, depending on the
underlying diseases.
systemic steroids did not lead to regression of the vessels. Our
results show that fine-needle feeder vessel coagulation
combined with topical bevacizumab application leads to
a statistically significant regression of mature corneal vascu-
larization. Regression of established vessels occurred to
a different degree in each patient. Five of 16 patients required
a second treatment. Two of these patients nonetheless showed
continuous progression of corneal NV. There are several
possible explanations for these variations. First, we had an
inhomogeneous study group composed of different underly-
ing etiologies for corneal NV (Fig. 1). Second, the remaining
blood vessels that were not coagulated may proliferate even
more. Third, the dose applied in this study may not have been
sufficient to antagonize VEGF because of the strong intensity
of the angiogenic stimulus caused by cautery and the under-
lying disease. The intensity of the angiogenic stimulus could
be different even between patients with similar corneal dis-
ease. Bevacizumab eye drops alone may not achieve suffi-
cient tissue levels of VEGF inhibition. Fourth, cytokines
other than VEGF (such as fibroblast growth factor and trans-
forming growth factor-a), which can induce corneal vascu-
larization, were not antagonized by bevacizumab.13,18 It
seems that patients with mature corneal NV due to herpetic
keratitis would benefit more from this new therapy regimen
than patients with other underlying corneal diseases.
Additional subconjunctival bevacizumab injection at
the time of cautery on top of anti-VEGF eye drops leads to
a stronger regression of corneal vascularization. This suggests
that in future controlled trials cautery should be combined
with subconjunctival anti-VEGFs in addition to anti-VEGF
eye drops.
Ó 2012 Lippincott Williams & Wilkins
Cornea  Volume 31, Number 8, August 2012
FIGURE 4. Additional subconjunctival bevacizumab injection
on top of anti-VEGF eye drops leads to significantly greater
reductions of the vascularized corneal area caused by cautery.
Our results show a nonsignificant improvement of the
visual acuity. We could not demonstrate that fine-needle
feeder vessel coagulation combined with subconjunctival
bevacizumab injection was beneficial toward improving
visual acuity, despite significantly better results in the
reduction of the vascularized corneal area. The only patient
with decreased visual acuity in our study had a large lipid
keratopathy due to herpetic keratitis. This patient was treated
twice with feeder vessel coagulation and subconjunctival
bevacizumab. Despite successful treatment of the corneal
vessels, the patient showed an enlarged area of lipid
keratopathy. All other patients with corneal scarring showed
decreased corneal opacity after treatment.
In our study—using cautery combined with anti-
VEGFs—we achieved occlusion of all vessels in 11 of 16 eyes
(68.8 %). Pillai et al8 showed occlusion of all vessels in 8 of
14 eyes (57.1 %) with cautery alone. The difference between
FIGURE 5. A, Corneal NV due to herpetic keratitis. B, Two months after fine-needle vessel coagulation and additional topical
bevacizumab application, there is a remaining scar without perfused vessels. C, 15 months later, the scar has remarkably cleared
up. No signs of reperfusion are present.
Ó 2012 Lippincott Williams & Wilkins
Angioregressive Pretreatment Prior to Keratoplasty
FIGURE 6. Development of BCVA after cautery and anti-VEGF
therapy in patients with mature corneal NV. Preoperative and
postoperative BCVA (logMAR) of all patients is shown (Wil-
coxon matched pairs signed rank test; P = 0.08).
the studies may be because of the use of additional topical
anti-VEGFs in our study. An exact comparison of the 2 studies
is difficult because of different methods of morphometry of
vascularized corneal areas used and different patient baseline
values.
Limitations of our study are the lack of a control group
and the small patient number. Our past experience showed
that patients with mature corneal vessels, who were treated
with bevacizumab eye drops alone, showed only partial
regression of the vessels and the vessel diameter (data not
shown). The reason seems to be that mature corneal vessels
do not significantly depend on VEGF anymore.7
In summary, our results suggest that fine-needle
diathermy combined with topical bevacizumab treatment
seems to be a relatively safe and well-tolerated option for
the treatment of established mature corneal NV. This may not
only improve corneal transparency but also “preconditions”
such a cornea for future keratoplasty.19,20 Compelling clinical
and experimental evidence suggests that preexisting corneal
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Koenig et al
NV is the most significant risk factor for graft rejection after
subsequent penetrating keratoplasty.19 Further controlled pro-
spective randomized trials are necessary to learn more about
the long-term safety, tolerability, and efficiency of this ther-
apeutic regimen. In addition, we need to study whether anti-
angiogenic preconditioning using the described approach
improves subsequent graft survival in patients with vascular-
ized high(er) risk corneas, as is the case in animal models of
corneal transplantation.20
REFERENCES
1. Chang JH, Gabison EE, Kato T, et al. Corneal neovascularization. Curr
Opin Ophthalmol. 2001;12:242–249.
2. Bock F, Koenig Y, Dietrich T, et al. Inhibition of angiogenesis in the
anterior chamber of the eye. Ophthalmologe. 2007;104:336–344.
3. Cursiefen C, Chen L, Saint-Geniez M, et al. Nonvascular VEGF receptor
3 expression by corneal epithelium maintains avascularity and vision.
Proc Natl Acad Sci U S A. 2006;103:11405–11410.
4. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature.
2000;407:249–257.
5. Aiello LP, Avery RL, Arrigg PG, et al. Vascular endothelial growth
factor in ocular fluid of patients with diabetic retinopathy and other
retinal disorders. N Engl J Med. 1994;331:1480–1487.
6. Koenig Y, Bock F, Horn F, et al. Short- and long-term safety profile and
efficacy of topical bevacizumab (Avastin) eye drops against corneal
neovascularization. Graefes Arch Clin Exp Ophthalmol. 2009;247:
1375–1382.
7. Cursiefen C, Hofmann-Rummelt C, Küchle M, et al. Pericyte recruitment
in human corneal angiogenesis: an ultrastructural study with clinicopath-
ological correlation. Br J Opththalmol. 2003;87:101–106.
8. Pillai CT, Dua HS, Hossain P. Fine needle diathermy occlusion of cor-
neal vessels. Invest Ophthalmol Vis Sci. 2000;41:2148–2153.
892 | www.corneajrnl.com
Cornea  Volume 31, Number 8, August 2012
9. Junghans BM, Collin HB. The limbal vascular response to corneal injury.
An autoradiographic study. Cornea. 1989;8:141–149.
10. Bock F, Koenig Y, Kruse F, et al. Bevacizumab (Avastin) eye drops
inhibit corneal neovascularization. Graefes Arch Clin Exp Ophthalmol.
2008;246:281–284.
11. Bock F, Onderka J, Hos D, et al. Improved semiautomatic method for
morphometry of angiogenesis and lymphangiogenesis in corneal flat-
mounts. Exp Eye Res. 2008;87:462–470.
12. Philipp W, Speicher L, Humpel C. Expression of vascular endothelial
growth factor and its receptors in inflamed and vascularized human cor-
neas. Invest Ophthalmol Vis Sci. 2000;41:2514–2522.
13. Cursiefen C, Rummelt C, Küchle M. Immunohistochemical localization
of vascular endothelial growth factor, transforming growth factor alpha,
and transforming growth factor beta1 in human corneas with neovascu-
larization. Cornea. 2000;19:526–533.
14. Dastjerdi MH, Al-Arfaj KM, Nallasamy N, et al. Topical bevacizumab in
the treatment of corneal neovascularisation: results of a prospective,
open-label, noncomparative study. Arch Ophthalmol. 2009;127:
381–389.
15. Bahar I, Kaiserman I, McAllum P, et al. Subconjunctival bevacizu-
mab injection for corneal neovascularization. Cornea. 2008;27:
142–147.
16. Fossarello M, Peiretti E, Zucca I, et al. Photodynamic therapy of corneal
neovascularization with verteporfin. Cornea. 2003;22:485–488.
17. Gerten G. Bevacizumab (avastin) and argon laser to treat neovasculari-
zation in corneal transplant surgery. Cornea. 2008;27:1195–1199.
18. Soubrane G, Jerdan J, Karpouzas I, et al. Binding of basic fibroblast
growth factor to normal and neovascularized rabbit cornea. Invest Oph-
thalmol Vis Sci. 1990;31:323–333.
19. Bachmann B, Taylor RS, Cursiefen C. Corneal neovascularization as a
risk factor for graft failure and rejection after keratoplasty: an evidence-
based meta-analysis. Ophthalmology. 2010;117:1300–1305.
20. Dietrich T, Bock F, Yuen D, et al. Cutting edge: lymphatic vessels, not
blood vessels, primarily mediate immune rejections after transplantation.
J Immunol. 2010;184:535–539.
Ó 2012 Lippincott Williams & Wilkins