November 2002
Volume 43, Issue 11
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Retina  |   November 2002
Subconjunctival Administration of Bucillamine Suppresses Choroidal Neovascularization in Rat
Author Affiliations
  • Yasuo Yanagi
    From the Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan; the
    Department of Regenerative Medicine, Research Institute, International Medical Center of Japan, Tokyo, Japan; and the
  • Yasuhiro Tamaki
    From the Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan; the
  • Ryo Obata
    From the Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan; the
  • Kimimasa Muranaka
    From the Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan; the
  • Noriko Homma
    From the Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan; the
  • Hidehito Matsuoka
    Ophthalmic Research Division, Santen Pharmaceutical Co., Ltd., Nara Research and Development Center, Ikoma-shi, Japan.
  • Hidetoshi Mano
    Ophthalmic Research Division, Santen Pharmaceutical Co., Ltd., Nara Research and Development Center, Ikoma-shi, Japan.
Investigative Ophthalmology & Visual Science November 2002, Vol.43, 3495-3499. doi:
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      Yasuo Yanagi, Yasuhiro Tamaki, Ryo Obata, Kimimasa Muranaka, Noriko Homma, Hidehito Matsuoka, Hidetoshi Mano; Subconjunctival Administration of Bucillamine Suppresses Choroidal Neovascularization in Rat. Invest. Ophthalmol. Vis. Sci. 2002;43(11):3495-3499.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Bucillamine is an antirheumatic drug with antiangiogenic properties that is currently used in clinical practice. Because bucillamine inhibits the production of VEGF, it is possible that this drug may inhibit choroidal neovascularization (CNV). Thus, the effect of bucillamine on the eyes of rats with experimental CNV was investigated in vivo by subconjunctival injection or oral intake.

methods. CNV was induced in rat eyes by diode laser photocoagulation. The intensity of fluorescein leakage from the photocoagulated lesions was studied 7 and 14 days after photocoagulation. The areas of CNV lesions were measured histologically and studied immunohistochemically at days 4, 7, and 14. In addition, the concentration of the drug in ocular tissue and blood was measured by high-performance liquid chromatography–tandem mass spectrometry after the drug was delivered orally or subconjunctivally.

results. After subconjunctival injection, fluorescein leakage from the CNV lesions decreased significantly compared with the control eyes throughout the study period. Histologic and immunohistochemical analyses 4, 7, and 14 days after photocoagulation demonstrated that the average size of the CNV lesions was reduced in the bucillamine-treated eyes compared with the control eyes. Subconjunctival injection maximized the ocular drug concentration while minimizing the blood concentration of the drug compared with oral intake.

conclusions. Subconjunctival injection of bucillamine significantly reduced the leakage and size of experimental CNV. These results suggest that bucillamine may be beneficial in treating CNV and that further studies can be considered to evaluate this possibility.

Exudative age-related macular degeneration due to choroidal neovascularization (CNV) is one of the two leading causes of visual loss in developed countries. 1 Because effective treatments have been established for only a limited number of patients, much effort has been directed toward understanding the pathogenesis of CNV and inhibiting its development. 2 3 Similar to the angiogenesis occurring in diseases such as malignant tumors and rheumatoid arthritis, the development of CNV is believed to start with the destruction of the basement membrane by a protease, followed by vascular endothelial cell migration and proliferation and tubular formation. 4 Laboratory studies have demonstrated that many factors are involved in these processes. Available evidence suggests that vascular endothelial growth factor (VEGF) is one of the most important factors affecting these processes. 5 6 7 8 9 10 11 12 An upregulation in the production of VEGF is observed in human CNV 5 and was also demonstrated in a model of laser photocoagulation–induced CNV. 6 7 8 Moreover, the administration of VEGF promotes development of CNV, 9 10 11 whereas inhibition of the VEGF signal leads to its suppression. 12 Therefore, a compound with the potential to block the VEGF signal may inhibit development of CNV. 
Bucillamine is a disease-modifying antirheumatic drug that has been in clinical use for more than 10 years. Although bucillamine is thought to act by immunomodulation, 13 14 15 recent studies in vitro have demonstrated that it inhibits production of VEGF, 16 17 18 whereas studies in vivo have demonstrated that its administration lowers the plasma concentration of VEGF. 16 18 Notably, patients who are treated for rheumatologic diseases with this drug demonstrate reduced levels of VEGF compared with levels before the treatment began. 19 These observations raise the possibility that bucillamine inhibits development of CNV. 
Concomitantly, the drug concentration in retina, choroid-sclera, and blood was measured after the drug was delivered orally or subconjunctivally. 
Materials and Methods
Animals
Female Brown Norway (BN) rats weighing between 200 and 250 g were obtained from CLEA Japan (Tokyo, Japan). All experiments were conducted in accordance with the Animal Care and Use Committee and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Experimental CNV
General anesthesia was induced with an intraperitoneal injection (1000 μL/kg) of a mixture (5:1) of ketamine hydrochloride (Ketalar; Sankyo, Tokyo, Japan) and xylazine hydrochloride (Celactal; Bayer, Tokyo, Japan) or by inhalation of diethyl ether. The pupil was dilated with one drop of 0.5% tropicamide (Mydrin M; Santen Pharmaceutical, Osaka, Japan) for photocoagulation. Experimental CNV was created as described elsewhere 19 with minor modification. Six laser photocoagulations were applied to each eye between the major retinal vessels around the optic disc with a diode laser photocoagulator (DC-3000; Nidek, Osaka, Japan) and a slit lamp delivery system (SL150; Topcon, Tokyo, Japan) at a spot size of 75 μm, duration of 0.1 second, and intensity of 100 mW. 
Twenty-four BN rats subjected to laser photocoagulation were injected subconjunctivally with 2.5 or 5 mg bucillamine diluted in phosphate-buffered saline (PBS; 0.14 M NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and 1.5 mM KH2PO4 [pH 7.5]) per eye; or PBS alone as a control administered through a 30-gauge needle under a microscope (SZ1045; Olympus, Tokyo, Japan); or 100 or 300 mg/kg bucillamine orally administered in 1% methylcellulose or 1% methylcellulose alone as a control. Each drug was administered just after the photocoagulation (on day 0) and once daily thereafter. 
Grading of CNV with Fluorescein Angiography
At days 7 and 14, the lesions were studied by fluorescein angiography (FA), as described elsewhere. 20 The intensity of staining in late-phase FA (100–150 seconds after fluorescein injection) was scored as follows in a masked manner: 0, no staining; 1, slight leakage; 2, moderate leakage; 3, strong leakage (Fig. 1)
To examine the fluorescein intensity of bucillamine itself, 1 mL rat serum was mixed with 10 μL of a sodium fluorescein solution (100 mg/L) in combination with either 5 μL PBS or PBS with various quantities of drug. After incubation at room temperature for 5 minutes, the intensity of the leakage was measured in a fluorescence spectrophotometer with an excitation wavelength of 490 nm and an emission wavelength of 580 nm. 
Histologic Analysis
Histologic analyses were performed on at least six eyes from the bucillamine-treated group (5 mg/eye subconjunctival administration) and control group at days 4, 7, and 14. Briefly, the rats were killed with an overdose of pentobarbital sodium, and the eyes were immediately enucleated and prepared for light microscopy by immersing them in PBS containing 4% paraformaldehyde (PFA) for 12 hours. The eyes were then transferred into 70% ethanol and processed for paraffin embedding. Once embedded, 4.0-μm sections of tissue were prepared for staining with hematoxylin and eosin (H&E) or immunostaining. To measure the area of CNV, an effort was made to section the entire lesion serially. Only lesions in which good serial sections were obtained through the center of the CNV were used in the analysis. Lesions were excluded based solely on an inability to obtain accurate measurement because of poor-quality sections. To determine the size of a lesion, sections were examined with a microscope at ×400 magnification (BX51; Olympus). For each retinal sample, approximately 60 sections were examined and a lesion that contained its largest area in consecutive serial sections was chosen in each sample, similar to previous studies, with the observer masked as to the treatment. 12 21 Images were taken with a charge-coupled device camera (CCD DP50; Olympus) and the data were imported into a computer (Macintosh; Apple Computer, Cupertino, CA) using a software system (Studio Lite; produced by Raining Data Software, Irvine, CA, in collaboration with Vencor Software, Wingham, Ontario, Canada), and the area of CNV in the subretinal space in the H&E-stained tissue was outlined with the computer mouse and measured using NIH Image (developed at the National Institutes of Health, Bethesda, MD, and available in the public domain at http://rsb.info.nih.gov/nih-image/). 
For immunohistochemistry, slides were incubated in blocking solution (PBS containing 0.1% BSA and 2% calf serum) for 30 minutes, followed by incubation with mouse anti-rat CD31 monoclonal antibody (platelet–endothelial cell adhesion molecule [PECAM]-1; PharMingen, San Diego CA) at a dilution of 1:100 in the blocking solution for 16 hours. Green fluorescent dye (Alexa 488)–conjugated anti-mouse secondary antibody (Molecular Probes, Eugene, OR) was used at a dilution of 1:800 in the blocking solution. Images were captured with a confocal microscope (LSM510; Carl Zeiss, Thornwood, NY). 
Measurement of the Concentration of Bucillamine in Ocular Tissues and Blood
On day 14, the rats were killed with an overdose injection of pentobarbital sodium 30 minutes after the drug was administered, and blood samples were collected. A 0.1 mL volume of blood was added into a glass test tube containing 0.2 mL 0.1 M Tris-HCl buffer (pH 9.1, 5 mM EDTA and 2Na). A 0.4 mL volume of 10% methyl acrylate (MA) in acetonitrile was immediately added to the tube. The eyes were enucleated and after a 3-hour fixation in 4% PFA at room temperature, the anterior segment and vitreous were removed, and the choroid together with the sclera and retina were collected separately. Because it is technically difficult to dissect the choroid from the sclera, the drug concentrations in the choroid and sclera were measured together. The choroid-sclera and retina were homogenized in saline. The lipids were transferred into a glass test tube, to which 0.2 mL 0.1 M Tris-HCl buffer and 0.4 mL 10% MA were immediately added. 
The sample mixture was vortexed and left at room temperature for 30 minutes. The mixture was deproteinized with acetone and the precipitate was removed by centrifugation. The supernatant was transferred into a glass test tube and evaporated to dryness. The residue was dissolved in 1 mL of 0.1 M HCl and applied to a solid-phase extraction column (30 mg/1 mL; HLB; Oasis, Glendora, CA). The column was washed with water and eluted with methanol. The eluent was evaporated to dryness, and the residue was redissolved in 0.1 mL methanol and 50 mM acetic acid (1:1 vol/vol). 
The concentrations of bucillamine in samples were measured by high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) with an HPLC system (1100; Hewlett-Packard, Palo Alto, CA) and a triple-stage quadruple mass spectrometer (TSQ; ThermoQuest, San Jose, CA). Separation was achieved on a 150 × 2.0-mm interior diameter column (HG-5 Develosil; Nomura, Seto, Japan); operated at 40°C and 0.2 mL/min, using a mobile phase consisting of methanol and 50 mM acetic acid (1:1, vol/vol). Quantitative analysis was performed by selected reaction monitoring, in MS/MS positive ions, detecting at m/z 394 to 308. The protein content was determined by the method of Lowry et al. 22  
Statistical Analysis
Data were analyzed by Mann-Whitney test. P < 0.05 was considered statistically significant. 
Results
Effect of Subconjunctival Administration of Bucillamine
With the use of a diode laser under our experimental conditions, 78% (33/42) and 86% (36/42) of photocoagulation sites showed development of CNV, 7 and 14 days after photocoagulation, respectively, as characterized by leakage detected with FA. Histologic analysis confirmed damage to Bruch’s membrane and formation of neovascularization extending from choroid to subretinal space in lesions that showed hyperfluorescence, similar to previous studies. Because we found that bucillamine itself at concentrations up to 1000 μg/mL does not affect fluorescein intensity, as analyzed by spectrophotometer (data not shown), and presumably does not confound the grading by FA, the photocoagulated lesions were analyzed by FA. At days 7 and 14, FA revealed that rats treated with a subconjunctival injection of bucillamine showed a significant decrease in leakage as judged from the leakage score (Figs. 2A 2B) compared with control rats, suggesting the subconjunctival administration of bucillamine effectively suppresses CNV. Oral intake of bucillamine also had a tendency to suppress CNV at day 14 (Figs. 2C 2D) , although the effects were not significant. FA revealed no apparent abnormalities in normal retinal blood vessels (data not shown). 
Histologic Analysis of CNV
To confirm whether the decrease in leakage in FA from CNV by subconjunctival injection of bucillamine is actually a consequence of the suppression of CNV, we analyzed histologic sections of CNV by quantitative morphometric assessment of the lesions at days 4, 7, and 14. The results revealed that the average size of lesions in the bucillamine-treated eyes was reduced significantly compared with those in the control at day 4 (0.0127 ± 0.0019 and 0.0051 ± 0.0007 mm2 in the control and bucillamine-treated groups, respectively; P = 0.02; Figs. 3A 3D 3G ), day 7 (0.0211 ± 0.0041 and 0.0060 ± 0.0007 mm2 in the control and bucillamine-treated groups, respectively; P = 0.02; Figs. 3B 3E 3H ), and day 14 (0.0234 ± 0.0051 and 0.0049 ± 0.0013 mm2 in the control and bucillamine-treated groups, respectively; P = 0.01; Figs. 3C 3F 3I ), confirming the suppressive effect of bucillamine on CNV. Moreover, we found that bucillamine-treated lesions contained few vascular channels and RPE stromal cells at day 4 (Fig. 3D) , day 7 (Fig. 3E) , and day 14 (Fig. 3F) . Thus, we analyzed the lesions immunohistochemically with an antibody against CD31 (PECAM), a marker of vascular endothelial cells. In control CNV lesions, we found immunoreactive vascular endothelial cells in most of the lesions on days 4, 7, and 14 (Figs. 4A 4C , and data not shown). In contrast, we found few immunoreactive vascular endothelial cells on days 4, 7, or 14 in the bucillamine-treated group (Figs. 4B 4D , and data not shown). Outside photocoagulation-treated lesions on normal retinal blood vessels, light microscopic and immunohistochemical analyses revealed no apparent abnormalities (data not shown), similar to the results shown by FA. 
Measurement of Bucillamine Concentrations in Ocular Tissues and Blood
Concentrations of bucillamine were examined in ocular tissues (the choroid and sclera together and the retina) and in the blood of the rats after the drug was delivered orally or subconjunctivally. Local concentrations of bucillamine in retina (Table 1) and choroid-sclera were much higher after the drug was administered subconjunctivally than those after the drug was administered orally. In contrast, blood concentrations of bucillamine were much lower when the drug was delivered subconjunctivally than when it was administered orally. In both groups, rats that received a higher dose showed higher blood and tissue concentrations of bucillamine. 
Discussion
The rat CNV model used in the present study produced CNV at a slightly lower rate in diode laser photocoagulation, 23 but reproducibly and at a high rate compared with krypton laser photocoagulation, 8 24 25 26 as judged by leakage shown in FA. Although a laser-induced model is widely used for study of CNV, it is controversial whether it mimics the most common form of naturally occurring CNV in age-related macular degeneration. Rather, because photocoagulation creates CNV with relatively acute damage to Bruch’s membrane, it may mimic traumatic CNV. Although a difference exists between CNV due to age-related change and trauma, the essential process (that is, the break-up of the basement membrane, the migration and proliferation of vascular endothelial cells, and tubular formation) is similar, which validates the use of the laser-photocoagulation–induced model for evaluating the effect of a drug on CNV. 
In the present study, it was demonstrated that subconjunctival injection of bucillamine suppressed CNV, as demonstrated by FA and confirmed by histologic and immunohistochemical analyses. Under the conditions, FA and histologic experiments demonstrated no apparent abnormalities in the normal retina. Thus, this drug is likely to be beneficial in treating CNV when administered by subconjunctival injection. Moreover, subconjunctival injection would minimize the plasma concentration of the drug and avoid the adverse effects of orally administered bucillamine. Although our results indicate modest effects of bucillamine on CNV and the effectiveness of this drug for treatment of CNV in humans requires further investigation, we believe that bucillamine has great potential to treat CNV in humans, especially when used in combination with other drugs, because a previous study in vitro has demonstrated synergistic effects on suppression of VEGF when used in combination with other drugs. 18  
Previous studies in vitro have demonstrated that a concentration of 10−4 M is effective in inhibiting the production of VEGF. 17 However, the optimal concentration of bucillamine for inhibition of neovascularization in vivo has not been studied. We have shown that a mean concentration of 4,726 and 36,646 ng/mg protein in retina and choroid-sclera, respectively, was sufficient to suppress CNV in the 2.5-mg/eye subconjunctival administration group and that a mean concentration of 7,796 and 123,477 ng/mg protein in retina and choroid-sclera, respectively, was more potent in the 5-mg/eye subconjunctival administration group. The ocular concentration of bucillamine was approximately 1.6 × 10−6 M when the drug was administered by subconjunctival injection, whereas after oral intake of 300 mg/kg it was approximately 2.6 × 10−8 M, as measured using isotope-labeled bucillamine (Matsuoka H, unpublished results, 2000). Because our study showed the tendency but failed to demonstrate a suppressive effect after oral administration, it is likely that the concentration of bucillamine in the choroid must be higher than 10−6 M to suppress CNV effectively in vivo. 
We confirmed the suppressive effect of bucillamine on CNV by histologic analysis and found a reduction in CNV lesion size, vascular formation, and vascular cells in the treated eyes. These observations imply that the formation of vascular tubes in CNV is blocked by bucillamine in vivo. Considering the reduced size of CNV in the bucillamine-treated group compared with the control group, it is likely that the proliferation and/or migration of vascular endothelial cells is also inhibited in bucillamine-treated eyes. Given that VEGF production was found to be suppressed by bucillamine treatment in vitro and in vivo, 16 17 18 19 it is likely that bucillamine acts to inhibit the production of VEGF, although it is still possible that bucillamine suppresses CNV by modulating immune reaction, which is likely to be implicated in CNV growth at least in part. 26  
In conclusion, in our studies subconjunctival injection of bucillamine suppressed CNV. Although our findings failed to demonstrate that oral intake of bucillamine is effective in suppressing CNV, an epidemiologic study would clarify whether oral intake inhibits CNV in humans, in that bucillamine has been used to treat rheumatoid arthritis for more than 10 years. 
 
Figure 1.
 
Representative fluorescein angiograms taken after diode laser photocoagulation. The intensity of the fluorescein leakage from each lesion was graded in a masked manner. The angiographies were graded as follows: 0, no staining; 1, slightly stained; 2, moderately stained; 3, strongly stained.
Figure 1.
 
Representative fluorescein angiograms taken after diode laser photocoagulation. The intensity of the fluorescein leakage from each lesion was graded in a masked manner. The angiographies were graded as follows: 0, no staining; 1, slightly stained; 2, moderately stained; 3, strongly stained.
Figure 2.
 
Decreased FA leakage in bucillamine-treated rats. Effects of bucillamine administered by (A, B) subconjunctival injection and (C, D) oral intake on days 7 (A, C) and 14 (B, D). Data are the mean score; each circle shows the intensity score of fluorescein leakage. *P < 0.05, **P < 0.01 by Mann-Whitney test.
Figure 2.
 
Decreased FA leakage in bucillamine-treated rats. Effects of bucillamine administered by (A, B) subconjunctival injection and (C, D) oral intake on days 7 (A, C) and 14 (B, D). Data are the mean score; each circle shows the intensity score of fluorescein leakage. *P < 0.05, **P < 0.01 by Mann-Whitney test.
Figure 3.
 
Light microscopic analyses of CNV in bucillamine-treated eyes. (AC) Light microscopy of lesions from control rats at days 4 (A), 7 (B), and 14 (C). (DF) Light microscopy of lesions from bucillamine-treated rats at days 4 (D), 7 (E), and 14 (F). (GI) Statistical analysis of the area of the lesions. Data show the mean size of CNV lesions from six (G), six (H), and eight (I) eyes; error bars indicate SEM. Note that in all study periods there was a significant difference in CNV area between control and bucillamine-treated rats. *P < 0.05 by Mann-Whitney test.
Figure 3.
 
Light microscopic analyses of CNV in bucillamine-treated eyes. (AC) Light microscopy of lesions from control rats at days 4 (A), 7 (B), and 14 (C). (DF) Light microscopy of lesions from bucillamine-treated rats at days 4 (D), 7 (E), and 14 (F). (GI) Statistical analysis of the area of the lesions. Data show the mean size of CNV lesions from six (G), six (H), and eight (I) eyes; error bars indicate SEM. Note that in all study periods there was a significant difference in CNV area between control and bucillamine-treated rats. *P < 0.05 by Mann-Whitney test.
Figure 4.
 
Immunohistochemical analyses of CNV in bucillamine-treated eyes. Sections of lesions from (A, C) control and (B, D) bucillamine-treated rats on days 4 (A, B) and 7 (C, D) were immunostained with an antibody against CD31 (PECAM). Note the decreased number of immunoreactive vascular cells in lesions from bucillamine-treated rats. Results of immunohistochemical analyses at day 14 were similar to those at day 7.
Figure 4.
 
Immunohistochemical analyses of CNV in bucillamine-treated eyes. Sections of lesions from (A, C) control and (B, D) bucillamine-treated rats on days 4 (A, B) and 7 (C, D) were immunostained with an antibody against CD31 (PECAM). Note the decreased number of immunoreactive vascular cells in lesions from bucillamine-treated rats. Results of immunohistochemical analyses at day 14 were similar to those at day 7.
Table 1.
 
Concentration of Bucillamine in Choroid/Sclera, Retina, and Blood plasma in Rats Treated by Oral Intake or Subconjunctival Injection
Table 1.
 
Concentration of Bucillamine in Choroid/Sclera, Retina, and Blood plasma in Rats Treated by Oral Intake or Subconjunctival Injection
Group Dose n Choroid/Sclera (ng/mg Protein) Retina (ng/mg Protein) Blood (μg/mL)
SCI 2.5 mg/eye 2 36,646 4,726 2.9
5 mg/eye 4 123,477 ± 72,816 7,796 ± 3,925 9.8 ± 4.9
Oral 100 mg/kg 4 497 ± 198 84 ± 29 15.1 ± 2.9
300 mg/kg 3 1,970 ± 557 298 ± 125 58.5 ± 12.8
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Figure 1.
 
Representative fluorescein angiograms taken after diode laser photocoagulation. The intensity of the fluorescein leakage from each lesion was graded in a masked manner. The angiographies were graded as follows: 0, no staining; 1, slightly stained; 2, moderately stained; 3, strongly stained.
Figure 1.
 
Representative fluorescein angiograms taken after diode laser photocoagulation. The intensity of the fluorescein leakage from each lesion was graded in a masked manner. The angiographies were graded as follows: 0, no staining; 1, slightly stained; 2, moderately stained; 3, strongly stained.
Figure 2.
 
Decreased FA leakage in bucillamine-treated rats. Effects of bucillamine administered by (A, B) subconjunctival injection and (C, D) oral intake on days 7 (A, C) and 14 (B, D). Data are the mean score; each circle shows the intensity score of fluorescein leakage. *P < 0.05, **P < 0.01 by Mann-Whitney test.
Figure 2.
 
Decreased FA leakage in bucillamine-treated rats. Effects of bucillamine administered by (A, B) subconjunctival injection and (C, D) oral intake on days 7 (A, C) and 14 (B, D). Data are the mean score; each circle shows the intensity score of fluorescein leakage. *P < 0.05, **P < 0.01 by Mann-Whitney test.
Figure 3.
 
Light microscopic analyses of CNV in bucillamine-treated eyes. (AC) Light microscopy of lesions from control rats at days 4 (A), 7 (B), and 14 (C). (DF) Light microscopy of lesions from bucillamine-treated rats at days 4 (D), 7 (E), and 14 (F). (GI) Statistical analysis of the area of the lesions. Data show the mean size of CNV lesions from six (G), six (H), and eight (I) eyes; error bars indicate SEM. Note that in all study periods there was a significant difference in CNV area between control and bucillamine-treated rats. *P < 0.05 by Mann-Whitney test.
Figure 3.
 
Light microscopic analyses of CNV in bucillamine-treated eyes. (AC) Light microscopy of lesions from control rats at days 4 (A), 7 (B), and 14 (C). (DF) Light microscopy of lesions from bucillamine-treated rats at days 4 (D), 7 (E), and 14 (F). (GI) Statistical analysis of the area of the lesions. Data show the mean size of CNV lesions from six (G), six (H), and eight (I) eyes; error bars indicate SEM. Note that in all study periods there was a significant difference in CNV area between control and bucillamine-treated rats. *P < 0.05 by Mann-Whitney test.
Figure 4.
 
Immunohistochemical analyses of CNV in bucillamine-treated eyes. Sections of lesions from (A, C) control and (B, D) bucillamine-treated rats on days 4 (A, B) and 7 (C, D) were immunostained with an antibody against CD31 (PECAM). Note the decreased number of immunoreactive vascular cells in lesions from bucillamine-treated rats. Results of immunohistochemical analyses at day 14 were similar to those at day 7.
Figure 4.
 
Immunohistochemical analyses of CNV in bucillamine-treated eyes. Sections of lesions from (A, C) control and (B, D) bucillamine-treated rats on days 4 (A, B) and 7 (C, D) were immunostained with an antibody against CD31 (PECAM). Note the decreased number of immunoreactive vascular cells in lesions from bucillamine-treated rats. Results of immunohistochemical analyses at day 14 were similar to those at day 7.
Table 1.
 
Concentration of Bucillamine in Choroid/Sclera, Retina, and Blood plasma in Rats Treated by Oral Intake or Subconjunctival Injection
Table 1.
 
Concentration of Bucillamine in Choroid/Sclera, Retina, and Blood plasma in Rats Treated by Oral Intake or Subconjunctival Injection
Group Dose n Choroid/Sclera (ng/mg Protein) Retina (ng/mg Protein) Blood (μg/mL)
SCI 2.5 mg/eye 2 36,646 4,726 2.9
5 mg/eye 4 123,477 ± 72,816 7,796 ± 3,925 9.8 ± 4.9
Oral 100 mg/kg 4 497 ± 198 84 ± 29 15.1 ± 2.9
300 mg/kg 3 1,970 ± 557 298 ± 125 58.5 ± 12.8
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