Korean J Ophthalmol.  2011 Jun;25(3):189-195. 10.3341/kjo.2011.25.3.189.

Intravenously Administered Anti-recoverin Antibody Alone Does Not Pass through the Blood-Retinal Barrier

• Kim JH
• Kim JH
• Kim DH
• Park WY
• Kim KW
• Yu YS

• KMID: 1010027
• DOI: http://doi.org/10.3341/kjo.2011.25.3.189

Abstract

PURPOSE
Cancer-associated retinopathy is a paraneoplastic retinal degeneration which may primarily result from auto-immune mediated apoptosis. It has been hypothesized that high titer of auto-antibodies are able to cross the blood-retinal barrier (BRB) and to enter retinal cells to activate apoptotic pathway which has been already well-established. However, it still remains to be elucidated whether auto-antibodies could cross BRB in the retina. Herein, we demonstrated that intravenously administrated anti-recoverin antibodies could not pass through BRB and not lead to retinal cell death.
METHODS
Anti-recoverin antibody was intravenously injected to C57BL/6 mice, which were sacrificed 1 and 7 days to obtain eye. Vascular endothelial growth factor was intravitreally injected to induce BRB breakdown, which was confirmed by fluorescein angiography and western blotting for zonula occludens (ZO)-1, ZO-2 and occludin. To investigate the location of anti-recoverin antibody in the retina, immunofluorescein was performed. The retinal toxicity of intravenous anti-recoverin antibody was evaluated by histological examination and transferase-mediated dUTP nick-end labeling. Immunofluorescein staining for glial fibrillary acidic protein was done to address glial activation as well.
RESULTS
Intravenously administrated anti-recoverin antibodies were exclusively distributed on retinal vessels which were co-localized with CD31, and led to neither increase of glial fibrillary acidic protein expression, as an indicator of retinal stress, nor apoptotic retinal cell death. Moreover, even in the condition of vascular endothelial growth factor-induced BRB breakdown, anti-recoverin antibodies could not migrate across BRB and still remained on retinal vessels without retinal cytotoxicity.
CONCLUSIONS
Our results suggest that high titer of intravascular anti-recoverin antibodies could not penetrate into the retina by themselves, and BRB breakdown mediated by dysregulation of tight junction might not be sufficient to allow anti-recoverin antibodies to pass through BRB.

Keyword

Anti-recoverin antibody; Blood-retinal barrier; Cancer-associated retinopathy; Intravenous administration; Retina

MeSH Terms

Animals
Antibodies/*administration & dosage/*metabolism
Blood-Retinal Barrier/*metabolism
Cell Death/drug effects
Cells, Cultured
Female
Injections, Intravenous
Mice
Mice, Inbred C57BL
Recoverin/*immunology
Retina/cytology/drug effects
Retinal Vessels/metabolism

Figure

• 

  Fig. 1 Intravenously administrated anti-recoverin antibodies could not pass through the blood-retinal barrier. After intravenous administration of anti-recoverin antibodies (1 mg/mL, 400 µL), the mice were sacrificed and enucleated before and one and seven days after injection. (A) Immunofluorescence staining for anti-recoverin antibody and 4', 6-diamidino-2-phenolindole (DAPI) staining for nuclei were performed. (B) Immunofluorescence staining of anti-recoverin antibody and CD31 as an endothelial cell marker and DAPI staining for nuclei were performed. Arrow indicates merged immunofluorescence of anti-recoverin antibody and CD31. Asterisk indicates CD31-positive immunoreactivity without anti-recoverin antibody. Quantitative analysis for CD31- and anti-recoverin antibody-positive spots relative to all anti-recoverin antibody-positive spots was performed by measuring ten randomly-selected fields on each slide at 400×. Each point represents the mean (SD) of six independent experiments, *p < 0.05. Scale bar, 100 µm. GCL = ganglion cell layer; IPL=inner plexiform layer; INL = inner nuclear layer; OPL=outer plexiform layer; ONL = outer nuclear layer; HPF=high power field.

• 

  Fig. 2 Intravenously administrated anti-recoverin antibodies never induced cytotoxicity in the retina. After intravenous administration of anti-recoverin antibodies (1 mg/mL, 400 µL), the mice were sacrificed and enucleated before and one and seven days after injection. (A) The apoptotic cells in the sagittal sections from 12 to 6 o/c through the cornea parallel to the optic nerve were assessed using a transferase-mediated dUTP nick-end labeling (TUNEL) assay. TUNEL-positive cells were measured in ten randomly-selected fields on each slide at 400x. Each point represents the mean (SD) of six independent experiments, **p > 0.05. (B) Immunofluorescence staining of glial fibrillary acidic protein (GFAP), an astrocyte marker, was performed. Scale bar, 100 µm. DAPI = 4', 6-diamidino-2-phenolindole; GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; HPF=high power field.

• 

  Fig. 3 Intravascular anti-recoverin antibodies could not pass through the blood-retinal barrier (BRB) in the case of BRB breakdown. (A) Fluorescein angiography using high molecular weight (500,000) fluorescein-conjugated dextran was performed to evaluate vascular endothelial growth factor (VEGF)-induced vascular leakage in the retina. One day following intravitreal injection of 20 ng/mL VEGF in 1 µL PBS or PBS only to mice, a whole mount retinal preparation was performed 1 hour post-perfusion with fluorescein-conjugated dextran. These experiments were repeated six times with similar results. Scale bar, 100 µm. (B) Human retinal microvascular endothelial cells were incubated for 12 hours with 20 ng/mL VEGF. Western blot analysis for zonula occludens (ZO)-1, ZO-2, and occludin was performed. β-Actin served as the loading control. Quantitative analysis was performed by measuring protein expression relative to that of the control. Each point represents the mean (SD) of three independent experiments. *p < 0.05. Figures were selected as representative data from three independent experiments. (C) Intravenous administration of anti-recoverin antibodies (1 mg/mL, 400 µL) was performed one day after intravitreal injection of 20 ng/mL VEGF. The mice were sacrificed and enucleated one day after anti-recoverin antibody injection. Immunofluorescence staining of anti-recoverin antibody and CD31 as an endothelial cell marker and 4', 6-diamidino-2-phenolindole (DAPI) staining for nuclei were performed. Arrow indicates merged immunofluorescence of anti-recoverin antibody and CD31. Quantitative analysis for CD31- and anti-recoverin antibody-positive spots relative to all anti-recoverin antibody-positive spots was performed by measuring ten randomly-selected fields on each slide at 400×. Each point represents the mean (SD) of six independent experiments, *p < 0.05. Scale bar, 100 µm. GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; HPF=high power field.

• 

  Fig. 4 Intravenously administrated anti-recoverin antibodies under blood-retinal barrier breakdown conditions never increased retinal cell death. Intravenous administration of anti-recoverin antibodies (1 mg/mL, 400 µL) was performed one day after intravitreal injection of 20 ng/mL vascular endothelial growth factor (VEGF). The mice were sacrificed and enucleated one and seven days after anti-recoverin antibody injection. The apoptotic cells in the sagittal sections from 12 to 6 o/c through the cornea parallel to the optic nerve were assessed using a transferase-mediated dUTP nick-end labeling (TUNEL) assay. TUNEL-positive cells were measured in ten randomly-selected fields on each slide at 400×. Each point represents the mean (SD) of six independent experiments, **p > 0.05. Scale bar, 100 µm. DAPI = 4', 6-diamidino-2-phenolindole; GCL = ganglion cell layer; INL = inner nuclear layer; ONL = outer nuclear layer; HPF=high power field.

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