December 1999
Volume 40, Issue 13
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Immunology and Microbiology  |   December 1999
Cancer-Associated Retinopathy Induced by Both Anti-Recoverin and Anti-hsc70 Antibodies In Vivo
Author Affiliations
  • Hiroshi Ohguro
    From the Department of Ophthalmology, Sapporo Medical University School of Medicine, Sapporo, Japan.
  • Kei–ichi Ogawa
    From the Department of Ophthalmology, Sapporo Medical University School of Medicine, Sapporo, Japan.
  • Tadao Maeda
    From the Department of Ophthalmology, Sapporo Medical University School of Medicine, Sapporo, Japan.
  • Akiko Maeda
    From the Department of Ophthalmology, Sapporo Medical University School of Medicine, Sapporo, Japan.
  • Ikuyo Maruyama
    From the Department of Ophthalmology, Sapporo Medical University School of Medicine, Sapporo, Japan.
Investigative Ophthalmology & Visual Science December 1999, Vol.40, 3160-3167. doi:
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      Hiroshi Ohguro, Kei–ichi Ogawa, Tadao Maeda, Akiko Maeda, Ikuyo Maruyama; Cancer-Associated Retinopathy Induced by Both Anti-Recoverin and Anti-hsc70 Antibodies In Vivo. Invest. Ophthalmol. Vis. Sci. 1999;40(13):3160-3167.

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Abstract

purpose. In a previous study, both recoverin and heat shock cognate protein 70 (hsc 70) were found as autoantigens recognized by sera from four patients with cancer-associated retinopathy (CAR). This observation suggested that autoimmune reactions against recoverin and hsc 70 might be involved together in the pathogenesis of CAR. The purpose of the present study is to investigate the effects of these autoantibodies on retinas in vivo.

methods. Functional and morphologic properties of the retinas were evaluated after anti-recoverin and/or anti-hsc 70 antibodies were intravitreously injected into Lewis rats’ eyes.

results. Responses in electroretinogram (ERG) of eyes penetrated with anti-hsc 70 antibody were comparable with the control, but those with anti-recoverin antibody were remarkably reduced during the 3-week period after the injection. Such anti-recoverin antibody–induced reduction was significantly enhanced by copenetration with anti-hsc 70 antibody. Immunofluorescence microscopy demonstrated that after intravitreal injection, anti-recoverin antibody penetrated toward the outer nuclear layer (ONL) and outer segments within 12 to 24 hours, and the presence of the antibody in the retina diminished during the next few days. Histopathology revealed significant thinning of the ONL and inner nuclear layer (INL) in the affected retina in comparison with the control. Throughout the ONL and INL, apoptotic cells were recognized by TdT-dUTP terminal nick-end labeling. The antibody-induced retinal dysfunction was effectively treated by administrations of either corticosteroid or cyclosporin A.

conclusions. These observations suggest that anti-recoverin– and anti-hsc 70 antibody–induced retinal dysfunction in Lewis rat is a good model to study the pathophysiology of CAR.

Avisual paraneoplastic syndrome called cancer-associated retinopathy (CAR) has been identified in patients with small cell carcinoma of lung and other malignant tumors. 1 2 3 4 5 6 7 These patients are clinically characterized with photopsia, progressive visual loss with a ring scotoma, attenuated retinal arterioles, and abnormalities of the a- and b-waves of the electroretinogram (ERG). 8 Histopathologic and immunologic observations revealed that in CAR, loss of photoreceptor cells may be caused by an autoimmune reaction against a photoreceptor-specific 23-kDa calcium-binding protein called recoverin. 9 10 Functionally, recoverin was found to play a major role in light and dark adaptation by regulating rhodopsin phosphorylation and dephosphorylation in a calcium-dependent manner. 11 12 Recently, expression of recoverin has been identified in the cancer cells of CAR patients, 13 14 and expression of the CAR antigen (recoverin) is induced by an intraperitoneal cultivation of small cell carcinoma. 15 16 17 These observations allowed us to speculate that aberrant expression of recoverin in cancer cells may trigger an autoimmune reaction. In addition, other retinal antigens including 65-kDa protein, 18 19 20 48-kDa protein, 8 enolase (46-kDa protein), 21 and neurofilament (58- to 62-, 145-, and 205-kDa proteins) 22 are recognized by some CAR patients’ sera. Among these retinal autoantigens, recoverin alone 8 23 24 or a combination of recoverin and 65-kDa protein 18 19 20 have most frequently been shown as the immunoreactive bands in western blot analysis in the previous reports. Most recently our group identified the 65-kDa protein as heat shock cognate protein 70 (hsc 70). 20 Therefore, we suggested that both anti-recoverin and anti-hsc 70 antibodies are involved in the pathogenesis of CAR. 
In the present study, to further investigate the pathologic roles of anti-recoverin and anti-hsc 70 antibodies in CAR, we injected these antibodies into the vitreous cavity of Lewis rats and performed histopathologic and electrophysiological characterization of the retinas. 
Materials and Methods
All experimental procedures were designed to conform to both the ARVO Statement for Use of Animals in Ophthalmic and Vision Research and our own institution’s guidelines. Unless otherwise stated, all procedures were performed at 4°C or on ice using ice-cold solutions. Prednisolone and cyclosporin A were generous gifts from Shionogi Co. and Novartis Pharma Inc., respectively. 
Antibodies
Anti-recoverin serum or anti-arrestin serum was obtained by immunization of purified recoverin 25 or arrestin 26 from fresh bovine retinas with complete adjuvant by the method described elsewhere. IgG of these sera was isolated by using a protein G Sepharose column chromatography using the protocol described by the manufacturer. For affinity purification, the IgG was applied through a column of recoverin- or arrestin-conjugated Sepharose, and IgG binding to the column was eluted by lowering the pH using 0.2 M glycine buffer, pH 2.5. An aliquot (1 ml each) was collected and mixed immediately with 0.1 ml of 1 M Tris buffer, pH 8.5. The purity and protein contents were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and spectrophotometry, respectively. Anti-rabbit hsc 70 serum was purchased from StressGen Biotechnologies Corp. (Sidney, British Columbia, Canada). The specificity and titers were examined by western blot analysis, using a bovine retinal–soluble fraction as described in our previous study, 20 before the antibody penetration study described below. All antibodies were diluted with phosphate-buffered saline (PBS) to adjust the IgG concentration at 1 mg/ml. 
Anesthesia of the Animals
In the present study, 8-week-old Lewis rats (approximately 250 g) reared in cyclic light conditions (12 hours on/12hours off) were used. For anesthesia induction, rats inhaled diethylether. Once unconscious, the animals were injected intramuscularly with a mixture of ketamine (80–125 mg/kg) and xylazine (9–12 mg/kg). Adequacy of the anesthesia was tested by tail-clamping, and supplemental doses of the mixture were administrated intramuscularly if needed. 
Vitreous Injection of Antibodies
Under anesthesia a total of 10 μl of PBS solution containing anti-recoverin IgG (5 μl), anti-hsc 70 serum (5 μl), anti-arrestin IgG (5 μl), or mixtures of them was administrated into the vitreous cavity of a rat eye. The injection was performed with a 26-gauge Hamilton micro-needle syringe through the sclera at a point 1 mm from the limbus to avoid puncture through the lens. Animals showing apparent traumatic changes after vitreous injection, such as cataract were excluded from the present study. After the surgery, a drop of 0.5% ofloxacin was administrated to avoid infection. 
Examinations by Slitlamp and Indirect Fundus Scopy
Under anesthesia, the anterior segment and fundus appearance of the animals’ eyes were carefully examined by a slitlamp and an indirect fundus scopy using a 78D preset lens, respectively, after the eyes were fully dilated by drops of 0.5% tropicamide. 
Electroretinography
The anesthetized animals were kept in dark adaptation for at least 1 hour in an electrically shielded room. The pupils were dilated with drops of 0.5% tropicamide. The scotopic ERG response was recorded with a contact electrode equipped with a suction apparatus to fit on the cornea (Kyoto contact lens Co.). A grounding electrode was placed on the ear. Responses evoked by white flashes (3.5 × 102 lux, 200-ms duration) were recorded by a Neuropack (MES-3102, Nihon Kohden, Tokyo, Japan). 
Light Microscopy
Anesthetized animals were transcardially perfused with a total 100 ml of 82 mM sodium phosphate buffer, pH 7.2, containing 4% paraformaldehyde. Enucleated eyes were embedded in paraffin and sectioned at 3-μm thickness, mounted on subbed slides, and dried. The sections were processed with hematoxylin-eosin staining after deparaffinization with graded ethanol and xylene solutions. Apoptotic cells in the retinal sections were detected by TUNEL (TdT-dUTP terminal nick-end labeling) stain using commercially available kits (Boehringer Mannheim, Mannheim, Germany) according to the protocol described by the manufacturer. 
Immunofluorescence Microscopy
Unfixed freshly dissected rat retinas were infiltrated with 30% sucrose in PBS at 4°C, cryosectioned at 10-μm thickness, mounted on subbed slides, air dried, and stored at −80°C before use. The sections were treated with ice-cold acetone for 10 minutes and air dried, and plastic rings were mounted around the sections to form incubation walls. The sections were incubated with goat anti-rabbit IgG labeled with Cy3 at 1:400 in PBS with 0.3% tween 20 at room temperature for 1 hour. The sections were then rinsed three times with PBS for 5 minutes and coverslipped in 90% glycerol in PBS containing 2% 1,4-diazabicyclo-(2,2,2)-octane. The sections were photographed using a Cy3 filter set. 
Statistical Analysis
Measurements of the retinal layers were compared between control and anti-recoverin– and anti-hsc 70 antibody–treated animals. Significant differences between groups were found using the post hoc test of Scheffé with a significance level set at P = 0.05 or 0.01. 
Drug Administration
Prednisolone (0.6 mg/kg/d) and cyclosporin A (10 mg/kg/d) dissolved in PBS and pure olive oil, respectively were each administrated by intramuscular injection. 
Results
We have identified both recoverin and hsc 70 as autoantigens recognized by sera from patients with CAR. 20 In the present study, to investigate the pathogenic effects of these autoantibodies on retinal cells, we injected affinity-purified antibodies against bovine recoverin, bovine arrestin, and rabbit hsc 70 into the vitreous cavity of Lewis rats. As a control, PBS was injected. The specificity and the titers of these antibodies were determined by a western blot analysis using a retinal soluble fraction (Fig. 1) . The specific labeling by anti-recoverin, anti-arrestin, and anti-hsc 70 were obtained by up to 1: 6000, 1: 6000, and 1: 4000 dilutions, respectively. After the injection, evaluations of retinal function and morphology were performed by slitlamp examination, indirect fundus scopy examination, electroretinogram (ERG), and light microscopy examination of the retinal sections. Examinations by slitlamp and fundus scopy detected no significant changes, such as retinal detachment, vitreoretinal hemorrhage, uveitis, or cataract in any animals without trauma after the injection. The numbers of rats used in the present study are summarized in Table 1
Figure 2 demonstrates typical ERG responses in animals at 3 weeks after the vitreous injections (experiment I). No changes in ERG were detected from eyes injected with anti-hsc 70 antibody compared with control (PBS injection) (n = 16–20 eyes for each experimental conditions). In contrast, significantly lower amplitudes of a- and b-waves in ERG were observed in eyes injected with anti-recoverin IgG. ERG responses were almost lost in eyes injected with both anti-recoverin and anti-hsc 70 antibodies. To exclude any unexpected effects on the response in case where mixtures of anti-recoverin and anti-hsc 70 antibodies were used, anti-arrestin and anti-hsc 70 antibodies were injected as an additional control. No significant ERG changes were observed in this control. 
Time course of the changes in the ERG responses (b-wave) after the injection are plotted in Figure 3 (changes in a-wave were almost parallel with those in b-wave) (n = 16–20 eyes for each experimental conditions) (experiment II). Control (PBS injection) and anti-hsc 70 antibody injection caused no effects until 5 weeks. In animals injected with anti-recoverin antibody, ERG responses were decreasing during the first 2 weeks and then reached plateau levels. Animals injected with both anti-recoverin and anti-hsc 70 antibodies were fully affected within 1 week after the injection. These data clearly indicated that anti-recoverin antibody directly caused retinal dysfunction, and this anti-recoverin antibody–induced retinal damage was enhanced and speeded up by the presence of anti-hsc 70 antibody. 
To clarify how these antibodies cause such retinal dysfunction, the distribution of anti-recoverin antibody within retina after the vitreous injection was studied immunocytochemically in frozen sections obtained at 3, 6, 12, and 24 hours and 3 and 6 days after the injection (experiment III). As shown in Figure 4 , the antibody was recognized within the inner parts of the retina at 3 hours, and then antibody localization shifted toward outer parts of the retina during 12 hours. During 12 to 24 hours, the antibody accumulated within both the outer nuclear layer (ONL) and photoreceptor layer, and thereafter the antibody diminished slowly from the retina within 6 days. Light microscopy of the retinal sections stained by hematoxylin-eosin revealed significant thinning of the ONL, inner nuclear layer (INL), and outer segment (OS) in the affected retina compared with control (Figs. 5A 5B , Table 2 ) (experiment IV). Among these layers, ONL was the most affected (Table 2) . However, inflammatory changes, such as destruction of the morphology and lymphocyte infiltrations, were not detected at all. In addition, TUNEL stain of the sections identified apoptotic cells throughout the ONL and INL (Fig. 5C) . Therefore, it is considered likely that apoptosis contributed to the anti-recoverin antibody–induced retinal dysfunction. 
To study the effects of corticosteroid or immunosuppressive agent, which are used frequently for treatment of human CAR patients, prednisolone or cyclosporin A was intramuscularly administrated to the affected animals (n = 10 eyes for each experimental conditions) everyday for 2 weeks after the injection of both anti-recoverin and anti-hsc 70 antibodies, as above (experiment V). As shown in Figure 6 , the initial decrease in the ERG responses, observed at 1 week after the vitreous injection, was relatively less in animals treated with prednisolone than in those without treatments. However, the responses in the prednisolone-treated animals gradually decreased, and no difference was observed between the two after 5 weeks. In contrast, administration of cyclosporin A did not effect on the initial retinal damage, but remarkable recovery of the ERG responses was noticed during the disease course. 
Discussion
CAR is believed to be caused by an autoimmune mechanism because of the presence of autoantibodies toward retinal components produced by some unknown processes. As retinal antigens recognized by CAR patients’ sera, recoverin (23-kDa protein), 8 23 24 65-kDa protein, 18 19 20 48-kDa protein, 8 enolase (46-kDa protein), 21 neurofilaments (58- to 62-kDa, 145-kDa, and 205-kDa proteins), 22 and others have been identified. Among these, recoverin, a Ca2+-binding regulatory protein specifically present in photoreceptor and bipolar cell, 27 is considered to be a major antigen involved in the pathogenesis of CAR because of the following reasons: (1) recoverin is a retina-specific protein, (2) recoverin is most frequently reported as an autoantigen in the previous case studies, and (3) recoverin is aberrantly expressed in cancer cells and their cell lines from CAR patients, 13 14 and its expression is induced by intraperitoneal cultivation of small cell carcinoma. 15 16 17 Our present study presented direct proof that anti-recoverin antibody causes a decrease of ERG responses in vivo, which is similar to the changes, observed in CAR patients. These observations suggested that serum anti-recoverin antibody is essentially required for developing the retinal degeneration. In fact, recently, Whitcup et al. 28 reported some interesting cases of CAR-like retinal degeneration, in which serum anti-recoverin was identified but malignant tumor was not apparent, and named this disease as “recoverin-associated retinopathy.” 
However, the pathologic roles of the other antigens are still unknown. In our recent study, 20 we found that 65-kDa protein, which was frequently identified as an autoantigen together with recoverin in patients with CAR, was identified as heat shock cognate protein 70 (hsc 70). Functionally, it is known that the heat shock protein 70 family acts as a chaperon of biological protection to suppress protein aggregation, denaturation, and misfolding under several stress conditions. 29 30 31 Because we believe that anti-recoverin antibody acts as a stress to photoreceptor cells, we speculate that anti-hsc 70 antibody may promote the anti-recoverin antibody–mediated retinal degeneration by blocking the chaperon functions of hsc 70. This speculation was proved by the present data that vitreous injection of anti-hsc 70 antibody did not affect the ERG responses, but significantly enhanced anti-recoverin antibody–induced changes in the responses. Similarly to CAR, serum autoantibodies against hsps have been identified in several autoimmune diseases, such as systemic lupus erythematosus (SLE), 32 33 rheumatoid arteritis (RA) 34 and mixed connective tissue disease. 35 Although the pathophysiological significance of the presence of the serum antibody to hsps is still unknown in these diseases, the above observations allowed us to speculate that a similar pathologic role to the autoimmune reaction toward hsp is involved. 
In terms of histopathologic changes observed in CAR patients, there has been little evidence of retinal inflammation, such as seen in patients with uveitis. This suggests that some noninflammatory mechanism is involved in the retinal degeneration. In retinal sections of rat treated with intravitreous injection of anti-recoverin and anti-hsc 70 antibodies, we found significant thinning of the retinal nuclear layers (ONL and INL) and outer segment layer, which is similar to the histopathologic changes observed in retinal sections from patients with CAR. Staining sections by TUNEL identified positively stained cells throughout the rat retina, suggesting that apoptosis may be primarily involved in the cell loss of the ONL and INL. Observations consistent with those were made by Adamus et al. 36 They injected monoclonal antibody against recoverin into vitreous of Lewis rats and found apoptosis of ONL and INL detected by TUNEL labeling, DNA fragmentation, and electron microscopic features. In addition, they also confirmed that anti-recoverin antibody–induced apoptotic cell death using a retinal cell culture system. 37 However, we still do not know how anti-recoverin and anti-hsc 70 antibodies bind with the target molecules and cause cell death by apoptotic processes, since both recoverin and hsc 70 are known to be present within cytosol. With regard to the antibody internalization, much experimental evidence has been reported in other paraneoplastic disorders 38 and autoimmune diseases. 39 Furthermore, Adamus et al. 37 claimed anti-recoverin antibody uptake by retinal cells in vitro resulted in apoptotic cell death. If this is possible, anti-recoverin and anti-hsc 70 antibodies may block the functions of recoverin, which regulates rhodopsin phosphorylation in a Ca2+-dependent manner, 11 12 and biological defense by hsc 70. In fact, recent observations have revealed that absence or abnormally high levels of rhodopsin phosphorylation are possible mechanisms of retinal degeneration in retinitis pigmentosa (RP). 40 41 42 Taken together, these observations suggest that abnormal regulation of rhodopsin phosphorylation may commonly be involved in the pathogenesis of photoreceptor degeneration in CAR and RP. 
In terms of localization of recoverin and histologic changes of anti-recoverin– treated retinas, they seemed to be inconsistent since recoverin is believed to localize mainly within photoreceptor outer segments. However, previous immunohistochemical studies have revealed that recoverin is localized within not only photoreceptor outer segments, but also in inner segments and synapse of photoreceptor and in bipolar cells. 27 Functionally, this allowed us to speculate that recoverin may be involved in significant roles other than the regulation of rhodopsin phosphorylation within these retinal cells. In fact, it was already known that calcium-binding regulatory proteins homologous with recoverin are widely distributed within the nervous system and may play significant roles in the calcium-signaling system. 43 Therefore, it was not surprising that anti-recoverin antibody–induced apoptosis within the ONL and INL. Alternatively, apoptosis within the ONL and INL may be a secondary event after the cell death of the photoreceptors. 
In terms of therapy of patients with CAR and other paraneoplastic syndromes, such as paraneoplastic cerebellar degeneration and Lambert–Eaton myasthenic syndrome, steroid administration, immunomodulation, and plasmapheresis have been clinically performed in conjunction with anti-neoplastic therapy. 44 45 46 For CAR, no definitive therapy has been established, although it has been reported that these above treatments may be effective in some patients. 3 5 6 8 20 47 Our results indicated that steroid and cyclosporin A were effective at the onset and during the course, respectively, of the anti-recoverin– and anti-hsc 70 antibody–induced retinal dysfunction. With regard to steroid, several immunologic effects have revealed, including reduction of the inflammatory response to immunologic processes and the prevention of the passage of antigen-antibody complexes through the basal membrane, 48 49 50 the latter of which seems likely to be the reason why steroids effect retinal dysfunction. Cyclosporin A has been extensively studied in both basic and clinical immunology for its unique immunosuppressive activities in selective suppression of T-cell functions. 51 52 53 This has been applied clinically for treatment of rejection in organ transplantation and in some autoimmune based diseases. In the present case, cyclosporin A was apparently effective on the antibody mediated retinal dysfunction. Although the precise pharmacological basis of this effect is unknown, it was suggested that local cell-mediated immunologic responses might also have contributed to the retinal damage. 
In conclusion, our present study clearly showed that both anti-recoverin and anti-hsc 70 antibodies directly cause CAR-like retinal dysfunction in vivo and that this antibody injection model is useful for understanding pathophysiology of CAR. 
 
Figure 1.
 
Western blot analysis of antibodies for in vivo administration. Affinity-purified anti-bovine recoverin IgG [lane 1, 1:2000 dilution (0.5 μg/ml)], affinity purified anti-bovine arrestin IgG [lane 2, 1:2000 dilution (0.5 μg/ml)] and anti-rabbit hsc 70 serum [lane 3, 1:2000 dilution (0.5μ g IgG/ml)] were tested with bovine retinal soluble extract.
Figure 1.
 
Western blot analysis of antibodies for in vivo administration. Affinity-purified anti-bovine recoverin IgG [lane 1, 1:2000 dilution (0.5 μg/ml)], affinity purified anti-bovine arrestin IgG [lane 2, 1:2000 dilution (0.5 μg/ml)] and anti-rabbit hsc 70 serum [lane 3, 1:2000 dilution (0.5μ g IgG/ml)] were tested with bovine retinal soluble extract.
Table 1.
 
Number of Rats Used in the ERG Study and the Histologic Analysis
Table 1.
 
Number of Rats Used in the ERG Study and the Histologic Analysis
Experiment Intravitreal Administration of Antibodies
PBS Anti-Recoverin Anti-hsc 70 Anti-Recoverin/Anti-hsc 70 Anti-Arrestin/Anti-hsc 70
I 8 (8) 8 (8) 8 (8) 10 (10) 8 (8)
II 8 (8) 10 (10) 8 (8) 10 (10) 8 (8)
IV 5 5
Experiment Time after Intravitreal Injection of Anti-Recoverin Antibody
3 h 6 h 12 h 24 h 3 d 6 d
III 2 2 2 2 2 2
Experiment Intramuscular Medication of Anti-Recoverin/Anti-hsc 70–Treated Rats
PBS Prednisolone Cyclosporin A
V 5 (5) 5 (3) 5 (4)
Figure 2.
 
Effects of intravitreal penetration of antibodies against recoverin, hsc 70, and/or arrestin on scotopic ERG. Either PBS, anti-hsc 70 serum (5 μg of IgG), anti-recoverin IgG (5 μg), a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-recoverin IgG (5 μg), or a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-arrestin IgG (5 μg) was injected intravitreously into Lewis rat eyes. Three weeks after the injection, a scotopic ERG was recorded. The details of ERG measurements are described in Materials and Method. The arrow indicates the timing of the light flash.
Figure 2.
 
Effects of intravitreal penetration of antibodies against recoverin, hsc 70, and/or arrestin on scotopic ERG. Either PBS, anti-hsc 70 serum (5 μg of IgG), anti-recoverin IgG (5 μg), a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-recoverin IgG (5 μg), or a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-arrestin IgG (5 μg) was injected intravitreously into Lewis rat eyes. Three weeks after the injection, a scotopic ERG was recorded. The details of ERG measurements are described in Materials and Method. The arrow indicates the timing of the light flash.
Figure 3.
 
Changes in scotopic ERG in the Lewis rats intravitreously penetrated with anti-recoverin IgG and/or anti-hsc 70 IgG. Either PBS (○), anti-hsc 70 serum (5 μg of IgG, ⋄), anti-recoverin IgG (5 μg,▵ ), or a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-recoverin IgG (5 μg) (□) was injected intravitreously into Lewis rat eyes. During the 5-week period after the operation, scotopic ERG was recorded once a week. The amplitudes of b-wave of the ERG were plotted. The details of ERG measurements are described in Materials and Methods.
Figure 3.
 
Changes in scotopic ERG in the Lewis rats intravitreously penetrated with anti-recoverin IgG and/or anti-hsc 70 IgG. Either PBS (○), anti-hsc 70 serum (5 μg of IgG, ⋄), anti-recoverin IgG (5 μg,▵ ), or a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-recoverin IgG (5 μg) (□) was injected intravitreously into Lewis rat eyes. During the 5-week period after the operation, scotopic ERG was recorded once a week. The amplitudes of b-wave of the ERG were plotted. The details of ERG measurements are described in Materials and Methods.
Figure 4.
 
Representative photomicrograph showing the penetration of anti-recoverin IgG into retina of Lewis rat. Each eye was injected intravitreously with affinity-purified anti-bovine recoverin rabbit IgG (5 μg). At 3, 6, 12, and 24 hours and 3 or 6 days after the operation, the eye was enucleated and fixed with 86 mM sodium phosphate buffer, pH 7.2, containing 4% paraformaldehyde for 10 minutes. Immediately after the fixation, frozen retinal sections were prepared and stored in −80°C. The presence of the antibody in the retina was visualized by anti-rabbit IgG labeled with Cy3 immunofluorescence. The details of the preparation of section and immunofluorescence staining are described in Materials and Methods. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment, Scale bar, 50μ m.
Figure 4.
 
Representative photomicrograph showing the penetration of anti-recoverin IgG into retina of Lewis rat. Each eye was injected intravitreously with affinity-purified anti-bovine recoverin rabbit IgG (5 μg). At 3, 6, 12, and 24 hours and 3 or 6 days after the operation, the eye was enucleated and fixed with 86 mM sodium phosphate buffer, pH 7.2, containing 4% paraformaldehyde for 10 minutes. Immediately after the fixation, frozen retinal sections were prepared and stored in −80°C. The presence of the antibody in the retina was visualized by anti-rabbit IgG labeled with Cy3 immunofluorescence. The details of the preparation of section and immunofluorescence staining are described in Materials and Methods. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment, Scale bar, 50μ m.
Figure 5.
 
Histopathologic changes in the retina of Lewis rats treated with anti-recoverin and anti-hsc 70 antibodies. Hematoxylin-eosine staining (A, B) or TUNEL staining (C) of retinal sections near the posterior pole from Lewis rat eyes, which were treated with PBS (A) or anti-recoverin and anti-hsc 70 antibodies (B and C, respectively). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment. Scale bar, 50 μm.
Figure 5.
 
Histopathologic changes in the retina of Lewis rats treated with anti-recoverin and anti-hsc 70 antibodies. Hematoxylin-eosine staining (A, B) or TUNEL staining (C) of retinal sections near the posterior pole from Lewis rat eyes, which were treated with PBS (A) or anti-recoverin and anti-hsc 70 antibodies (B and C, respectively). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment. Scale bar, 50 μm.
Table 2.
 
Thickness of the Retinal Layers
Table 2.
 
Thickness of the Retinal Layers
Layers (μm)
OS, † ONL* INL* IPL
Control (PBS) 45.3 ± 3.7 85.3 ± 2.3 55.1 ± 2.9 101.2 ± 3.9
Anti-recoverin/anti-hsc70 39.7 ± 2.2 66.9 ± 3.5 47.3 ± 2.2 100.0 ± 4.8
Antibodies
Figure 6.
 
Effects of corticosteroid or cyclosporin A administration on scotopic ERG in the Lewis rats intravitrously penetrated with anti-recoverin IgG and anti-hsc 70 serum. A mixture of anti-hsc 70 (5 μg of IgG) and anti-recoverin IgG (5 μg) was injected intravitreously into Lewis rat eyes, and these animals were intramuscularly administrated either PBS (○), prednisolone (0.6 mg/kg, ▵), or cyclosporin A (10 mg/kg, □) everyday during the next 2 weeks (marked by a thick line designated medication). During the 5-week period after the antibody penetration, scotopic ERG was recorded once a week. The amplitudes of b-wave of the ERG were plotted. The details of ERG measurements are described in Materials and Methods.
Figure 6.
 
Effects of corticosteroid or cyclosporin A administration on scotopic ERG in the Lewis rats intravitrously penetrated with anti-recoverin IgG and anti-hsc 70 serum. A mixture of anti-hsc 70 (5 μg of IgG) and anti-recoverin IgG (5 μg) was injected intravitreously into Lewis rat eyes, and these animals were intramuscularly administrated either PBS (○), prednisolone (0.6 mg/kg, ▵), or cyclosporin A (10 mg/kg, □) everyday during the next 2 weeks (marked by a thick line designated medication). During the 5-week period after the antibody penetration, scotopic ERG was recorded once a week. The amplitudes of b-wave of the ERG were plotted. The details of ERG measurements are described in Materials and Methods.
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Figure 1.
 
Western blot analysis of antibodies for in vivo administration. Affinity-purified anti-bovine recoverin IgG [lane 1, 1:2000 dilution (0.5 μg/ml)], affinity purified anti-bovine arrestin IgG [lane 2, 1:2000 dilution (0.5 μg/ml)] and anti-rabbit hsc 70 serum [lane 3, 1:2000 dilution (0.5μ g IgG/ml)] were tested with bovine retinal soluble extract.
Figure 1.
 
Western blot analysis of antibodies for in vivo administration. Affinity-purified anti-bovine recoverin IgG [lane 1, 1:2000 dilution (0.5 μg/ml)], affinity purified anti-bovine arrestin IgG [lane 2, 1:2000 dilution (0.5 μg/ml)] and anti-rabbit hsc 70 serum [lane 3, 1:2000 dilution (0.5μ g IgG/ml)] were tested with bovine retinal soluble extract.
Figure 2.
 
Effects of intravitreal penetration of antibodies against recoverin, hsc 70, and/or arrestin on scotopic ERG. Either PBS, anti-hsc 70 serum (5 μg of IgG), anti-recoverin IgG (5 μg), a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-recoverin IgG (5 μg), or a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-arrestin IgG (5 μg) was injected intravitreously into Lewis rat eyes. Three weeks after the injection, a scotopic ERG was recorded. The details of ERG measurements are described in Materials and Method. The arrow indicates the timing of the light flash.
Figure 2.
 
Effects of intravitreal penetration of antibodies against recoverin, hsc 70, and/or arrestin on scotopic ERG. Either PBS, anti-hsc 70 serum (5 μg of IgG), anti-recoverin IgG (5 μg), a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-recoverin IgG (5 μg), or a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-arrestin IgG (5 μg) was injected intravitreously into Lewis rat eyes. Three weeks after the injection, a scotopic ERG was recorded. The details of ERG measurements are described in Materials and Method. The arrow indicates the timing of the light flash.
Figure 3.
 
Changes in scotopic ERG in the Lewis rats intravitreously penetrated with anti-recoverin IgG and/or anti-hsc 70 IgG. Either PBS (○), anti-hsc 70 serum (5 μg of IgG, ⋄), anti-recoverin IgG (5 μg,▵ ), or a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-recoverin IgG (5 μg) (□) was injected intravitreously into Lewis rat eyes. During the 5-week period after the operation, scotopic ERG was recorded once a week. The amplitudes of b-wave of the ERG were plotted. The details of ERG measurements are described in Materials and Methods.
Figure 3.
 
Changes in scotopic ERG in the Lewis rats intravitreously penetrated with anti-recoverin IgG and/or anti-hsc 70 IgG. Either PBS (○), anti-hsc 70 serum (5 μg of IgG, ⋄), anti-recoverin IgG (5 μg,▵ ), or a mixture of anti-hsc 70 serum (5 μg of IgG) and anti-recoverin IgG (5 μg) (□) was injected intravitreously into Lewis rat eyes. During the 5-week period after the operation, scotopic ERG was recorded once a week. The amplitudes of b-wave of the ERG were plotted. The details of ERG measurements are described in Materials and Methods.
Figure 4.
 
Representative photomicrograph showing the penetration of anti-recoverin IgG into retina of Lewis rat. Each eye was injected intravitreously with affinity-purified anti-bovine recoverin rabbit IgG (5 μg). At 3, 6, 12, and 24 hours and 3 or 6 days after the operation, the eye was enucleated and fixed with 86 mM sodium phosphate buffer, pH 7.2, containing 4% paraformaldehyde for 10 minutes. Immediately after the fixation, frozen retinal sections were prepared and stored in −80°C. The presence of the antibody in the retina was visualized by anti-rabbit IgG labeled with Cy3 immunofluorescence. The details of the preparation of section and immunofluorescence staining are described in Materials and Methods. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment, Scale bar, 50μ m.
Figure 4.
 
Representative photomicrograph showing the penetration of anti-recoverin IgG into retina of Lewis rat. Each eye was injected intravitreously with affinity-purified anti-bovine recoverin rabbit IgG (5 μg). At 3, 6, 12, and 24 hours and 3 or 6 days after the operation, the eye was enucleated and fixed with 86 mM sodium phosphate buffer, pH 7.2, containing 4% paraformaldehyde for 10 minutes. Immediately after the fixation, frozen retinal sections were prepared and stored in −80°C. The presence of the antibody in the retina was visualized by anti-rabbit IgG labeled with Cy3 immunofluorescence. The details of the preparation of section and immunofluorescence staining are described in Materials and Methods. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment, Scale bar, 50μ m.
Figure 5.
 
Histopathologic changes in the retina of Lewis rats treated with anti-recoverin and anti-hsc 70 antibodies. Hematoxylin-eosine staining (A, B) or TUNEL staining (C) of retinal sections near the posterior pole from Lewis rat eyes, which were treated with PBS (A) or anti-recoverin and anti-hsc 70 antibodies (B and C, respectively). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment. Scale bar, 50 μm.
Figure 5.
 
Histopathologic changes in the retina of Lewis rats treated with anti-recoverin and anti-hsc 70 antibodies. Hematoxylin-eosine staining (A, B) or TUNEL staining (C) of retinal sections near the posterior pole from Lewis rat eyes, which were treated with PBS (A) or anti-recoverin and anti-hsc 70 antibodies (B and C, respectively). GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; OS, outer segment. Scale bar, 50 μm.
Figure 6.
 
Effects of corticosteroid or cyclosporin A administration on scotopic ERG in the Lewis rats intravitrously penetrated with anti-recoverin IgG and anti-hsc 70 serum. A mixture of anti-hsc 70 (5 μg of IgG) and anti-recoverin IgG (5 μg) was injected intravitreously into Lewis rat eyes, and these animals were intramuscularly administrated either PBS (○), prednisolone (0.6 mg/kg, ▵), or cyclosporin A (10 mg/kg, □) everyday during the next 2 weeks (marked by a thick line designated medication). During the 5-week period after the antibody penetration, scotopic ERG was recorded once a week. The amplitudes of b-wave of the ERG were plotted. The details of ERG measurements are described in Materials and Methods.
Figure 6.
 
Effects of corticosteroid or cyclosporin A administration on scotopic ERG in the Lewis rats intravitrously penetrated with anti-recoverin IgG and anti-hsc 70 serum. A mixture of anti-hsc 70 (5 μg of IgG) and anti-recoverin IgG (5 μg) was injected intravitreously into Lewis rat eyes, and these animals were intramuscularly administrated either PBS (○), prednisolone (0.6 mg/kg, ▵), or cyclosporin A (10 mg/kg, □) everyday during the next 2 weeks (marked by a thick line designated medication). During the 5-week period after the antibody penetration, scotopic ERG was recorded once a week. The amplitudes of b-wave of the ERG were plotted. The details of ERG measurements are described in Materials and Methods.
Table 1.
 
Number of Rats Used in the ERG Study and the Histologic Analysis
Table 1.
 
Number of Rats Used in the ERG Study and the Histologic Analysis
Experiment Intravitreal Administration of Antibodies
PBS Anti-Recoverin Anti-hsc 70 Anti-Recoverin/Anti-hsc 70 Anti-Arrestin/Anti-hsc 70
I 8 (8) 8 (8) 8 (8) 10 (10) 8 (8)
II 8 (8) 10 (10) 8 (8) 10 (10) 8 (8)
IV 5 5
Experiment Time after Intravitreal Injection of Anti-Recoverin Antibody
3 h 6 h 12 h 24 h 3 d 6 d
III 2 2 2 2 2 2
Experiment Intramuscular Medication of Anti-Recoverin/Anti-hsc 70–Treated Rats
PBS Prednisolone Cyclosporin A
V 5 (5) 5 (3) 5 (4)
Table 2.
 
Thickness of the Retinal Layers
Table 2.
 
Thickness of the Retinal Layers
Layers (μm)
OS, † ONL* INL* IPL
Control (PBS) 45.3 ± 3.7 85.3 ± 2.3 55.1 ± 2.9 101.2 ± 3.9
Anti-recoverin/anti-hsc70 39.7 ± 2.2 66.9 ± 3.5 47.3 ± 2.2 100.0 ± 4.8
Antibodies
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