1. Sang June Kim1, Ha Bum Lee1, Hungwong Tchah2 Hye Young Park1
Effect of intracameral injection of
bevacizumab on the rabbit corneal endothelium
1. Department of Ophthalmology
College of Hallym University
Hallym Medical Center Seoul, South Korea
2.Department of Ophthalmology
University of Ulsan College of Medicine
ASAN Medical Center Seoul, South Korea
Sang June Kim1, Ha Bum Lee1, Hungwong Tchah2
Hye Young Park1

2. Introduction
Bevacizumab(Avastin®, Genentech, San Francisco, California, USA)
recombinant humanized monoclonal Ig G1 antibody directed against
the vascular endothelial growth factor (VEGF)
Systemic use
first anti-angiogenic agent approved by the FDA for intravenous use
against metastatic colorectal cancer
Ophthalmic use
Avery RL, 2006; Iturralde D et al, 2006; Rosenfeld PJ et al, 2005;
Spaide RF & Fisher YL, 2006; Spaide RF et al, 2006 T
therapeutic benefits of intravitreal bevacizumab injection for various
Intraocular neovascular diseases
Grisanti S et al, 2006
intracameral bevacizumab injection resulted in encouraging preliminary
outcomes in neovascular glaucoma

3. Purpose Background Purpose
While studies on retinal cells provided information regarding safe and
effective doses of bevacizumab for intravitreal injection (Feiner L et al
, 2006;Inan UU et al, 2007;Luthra S et al, 2006;Spitzer MS et al, 2006)
, similar studies on corneal endothelial cells have not been undertaken.
Such information would assist in determining safe and effective concent
rations of intracameral bevacizumab injections. 
Purpose
The present study examined the effect of intracameral bevacizumab injection
on corneal endothelial cell morphology and viability in a rabbit model.

4. Materials and Methods Materials
Animals : 20 eyes of 10 New Zealand White rabbits (1.3∼1.7 kg)
* The animal experiments were reviewed and approved by the Institutional Animal
Care and Use Committee (IACUC) of the Asan Institute for Life Sciences, Asan
Medical Center. The committee abides by the Institute of Laboratory Animal
Resources (ILAR) guide.
Measurement
Specular microscopy (Topcon SP2000P)
: mean value from 3 consecutive measurement
Pachymetry (pachymetry; DGH-550 Pachette 2)
: mean value from 5 consecutive measurement
Slit lamp examination (Kowa, SL-15, portable slit lamp)

5. Materials and Methods Procedures and Examinations and Xylazine
Anesthesia : intramuscular injection of ketamine hydrochloride
and Xylazine
Grouping into 4 subgroups
* Avastin® , Genentech, San Francisco, California, USA
BSS® , Santen, Osaka, Japan
Post injection examination schedule
2 hours, 1 week, 4 weeks later after injection
Measurements
specular microscope, pachymeter and slit-lamp examination A B
Group1
Avatin® (0.125mg, 0.05cc)
BSS® (0.05cc)
Group2
Avastin® (0.25mg, 0.1cc)
BSS® (0.1cc)

6. Materials and Methods Tissue Preparation and Examinations
Corneal buttoning
4 weeks after injection, 2 cornea button was obtained from each subgroup
Stain methods
alizarin red (Sigma) and trypan blue (TCI) according to Taylor
and Hunt’s method (Spence DJ & Peyman GA, 1976; Taylor MJ & Hunt CJ, 1981).
Scanning Electron Microscopy(SEM)
method of Wang and Hu (Wang I-J & Hu F-R, 1997).
Statistical Analysis
Wilcoxon signed rank test
: compare data among groups
Mann-Whitney U-tests
: compare between each group
* Differences were considered statistically significant when P < 0.05.

7. Results
Portable slit lamp examination (Kowa, Sl-15, portable slit lamp) .
Slit-lamp microscopic examinations revealed no
apparent abnormalities in the corneas, anterior
chambers or lenses over the duration of the study
Specular Microscope (Topcon SP2000P)
For each individual subgroup, there was no significant change in any parameter during the study. In addition, there was no statistical difference in these parameter upon comparison between groups (See table 1, 2, 3)
Before
2 Hr
1 Wk
4 Wk
 1-A
2821±182
2833±146
2871±226
3079±140
 1-B
2933±387
2704±208
2890±99
3130±67
 2-A
3062±282
2952±324
2786±366
2922±300
 2-B
2916±404
2902±449
2822±413
3009±276
Table 1. Specular microscopic result - cell density (CD) (cells/mm2)
of each group before-and after injection (mean±SE)

8. Results
Before
2 Hr
1 Wk
4 Wk
  1-A
31±11
36±3
29±11
  1-B
34±4
30±4
28±7
22±7
  2-A
37±5
37±6
32±2
24±7
  2-B
32±7
28±3
23±5
Table 2. Specular microscopic result - coefficient of variation (CV) of
each group before-and after injection (mean±SE)
Before
2 Hr
1 Wk
4 Wk
   1-A
359±23
356±23
354±34
326±15
   1-B
379±82
373±28
347±12
319±7
   2-A
328±32
339±39
364±45
346±32
   2-B
348±42
357±53
362±54
334±30
Table 3. Specular microscopic result – average cell area (AVG) (unit/mm2)
of each group before-and after injection (mean±SE)

9. Results Pachymeter (pachymetry; DGH-550 Pachette 2)
The corneal thickness values for the 4 subgroups are shown in Table 4. These pachymetric data showed there were no significant differences between the subgroups.
Before
2 Hr
1 Wk
4 Wk
  1-A
328±17
359±35
336±20
335±20
   1-B
325±18
357±43
323±14
   2-A
325±17
361±29
321±35
331±38
   2-B
329±20
354±29
313±35
329±40
Table 4. Corneal thickness(㎛) of each group before-and injection (mean±SE)

10. Results
Histology
( Light Microscopic Image, magnification
X 200)
Histology
( Scanning Electron Micrographs of
corneal endothelial cells. )
Click here, you can see LM 5 images
Click here, you can see 4 SEM images B - 1
0.05mL BSS injection
Giant cell
(black arrow) B
0.05mL BSS injection
Untreated normal cornea
0.1mL BSS injection D C
0.1mL (2.5mg)
bevacizumab
injection
(black arrow)
0.05ml BSS injection
Giant cell (
0.05ml(1.25mg)
bevacizumab
injection
Giant cell
balck
arrow ) A
0.05mL (1.25mg)
bevacizumab
injection
0.1ml BSS injection
Giant cell
(black arrow)
0.1ml(2.5mg)
bevacizumab
injection
Giant cell
(black arrow)
While giant cells were observed in all subgourps, but cell borders and microvilli were well preserved. There was no evidence of abnormal endothelial cell formation such as blebbing or cellular border disintegration

11. Intracameral injection
Conclusion
Angiogenesis
associated wound healing
associated many ocular pathogenesis
Ex) NeoVascular Glaucoma , Diabetic Retinopathy
Corneal Neovascularization
Use of Bevacizumab on NVG
1. Intracameral injection 
2. Intravitreal injection 1
(Grisanti S et al, 2006)
Intracameral
bevacizumab injection
significantly inhibit iris
neovascularization. 2
(Aggio FB et al, 2007)
Intravitreal injection is
Also effective against iris
rubeosis, but endoph-
thalmitis and RPE tears
risk increased. 3
So,
Intracameral injection
is superior method,
if it is safe in terms of
corneal endothelium. VS

12. Conclusion
Previous experimental study about corneal toxicity of Bevacizumab
Click here !!
Yoeruek E et al(2007)
5.0mg/ml bevacizumab did not affect
cultured human corneal endothelial cell viability
Do you want our conclusion?
Click here again !!
Bevacizumab (up to 2.5 mg/0.1 mL) had no effect on
endothelial cell viability or morphology in the rabbit cornea.
Further studies are required to examine the effect of repeated
bevacizumab intracameral injections on corneal endothelium
such as would be used in clinical applications.