Immunolocalization of transforming growth factor β during wound repair in rat retina after laser photocoagulation

• Background: Scatter photocoagulation induces regression of retinal neovascularization, but the mechanism of its therapeutic effect is incompletely understood. To elucidate the mechanism of therapeutic effect of photocoagulation is the main focus of our research. We have already demonstrated basic fibroblast growth factor (bFGF) immunolocalization during retinal wound repair following laser photocoagulation. Transforming growth factor beta (TGF β) reportedly inhibits endothelial cell growth and bFGF-induced cell proliferation in vitro. In the present study, we evaluated the immunohistochemical localization of TGF-β1 and -β2 during wound repair in the rat retina following laser photocoagulation. • Methods: Krypton laser photocoagulation was performed on the eyes of pigmented rats. The eyes were then enucleated on day 1, 3, 7, 14, 28 or 56 following the photocoagulation and enrolled into the analysis of immunohistochemical localization of TGF-β1 and -β2. • Results: Immunoreactivity for TGF-β1 and -β2 was present in the ganglion cell layer and photoreceptor outer segments of the normal adult rat retina. The cytoplasm of RPE cells at the photocoagulated lesion showed intense TGF-β1 and -β2 immunoreactivity on day 3 after laser photocoagulation. Macrophages that migrated into the lesion lacked positive staining for TGF-β1 and -β2. TGF-β immunoreactivity in RPE cells continued to be upregulated for more than 1 month compared with that in normal RPE cells. Controls did not exhibit any positive staining. • Conclusion: An elevated expression of TGF-β immunoreactivity for a longer period of time than bFGF was observed in RPE cells at the photocoagulated lesion in vivo. In the late phase of retinal wound repair, TGF-β may inhibit cell proliferation induced by mitogens, introduce an end stage of cellular events, and induce extracellular matrix induction. This study was supported in part by The Science Research Promotion Fund of the Japan Private School Promotion Foundation (Dr. Uyama), and by a Grant-in-Aid for Developmental Scientific Research from the Ministry of Education, Science and Culture of the Japanese Government (Dr. Uyama)     

Immunolocalization of basic fibroblast growth factor during wound repair in rat retina after laser photocoagulation

Background: Basic fibroblast growth factor (bFGF) stimulates the mitogenesis of various cells and plays a key role in wound repair. We studied the immunohistochemical localization of bFGF during wound repair in the rat retina after laser photocoagulation. Methods: Krypton laser photocoagulation was performed on the eyes of pigmented rats. The eyes were enucleated on days 1, 3, 7, 14 and 28 after the photocoagulation, and the immunohistochemical localization of bFGF was assessed. Two different monoclonal antibodies and one polyclonal antibody against bFGF as first antibodies were used. Results: Marked immunoreactivity for bFGF was found in the ganglion cell layer, and weak immunoreactivity for bFGF was found in the retinal pigment epithelial (RPE) cells of the normal adult rat retina. On day 3 after laser photocoagulation, the nuclei and cytoplasm of proliferating RPE cells at the center of the photocoagulated lesion showed intense bFGF immunoreactivity. The nuclei of RPE cells around the lesion showed intense bFGF immunoreactivity. Macrophages that migrated into the lesion showed positive staining for bFGF. These immunoreactivity decreased with time. Controls (0.05 M Tris-HCl buffer, normal serum, or these same antibodies preabsorbed with bFGF) did not show positive staining. Conclusion: The finding of an elevated expression of bFGF immunoreactivity in the photocoagulated lesion suggests that bFGF may play a role in wound repair in the rat retina after laser photocoagulation.     

The effects of growth factors and conditioned media on the proliferation of human corneal epithelial cells and keratocytes

Background: As growth factors play an important role in epithelial wound repair, we evaluated the effect of exogenous growth factors in the presence and absence of corneal epithelial and keratocyte conditioned medium on human corneal epithelial cell and keratocyte proliferation. • Methods: Preconfluent cultures of human corneal epithelial cells or stromal keratocytes were exposed to varying concentrations of EGF, TGF-β or bFGF in the presence or absence of human corneal epithelial or stromal keratocyte conditioned medium. Cell numbers were determined after 48 h incubation. RIA and ELISA were used to quantify the levels of EGF, TGF-β and bFGF in conditioned media. • Results: EGF and bFGF increased, while TGF-β decreased, the proliferation of both cell types in a dose- dependent manner. Epithelial cell conditioned medium inhibited, and keratocyte conditioned medium stimulated, the proliferation of both cell types. The proliferative effects of EGF, TGF-β and bFGF in the presence of keratocyte conditioned medium were additive for both cell types. By contrast, the addition of exogenous growth factors was unable to overcome the inhibitory potential of epithelial conditioned medium. Both conditioned media contained significant levels of bFGF, but TGF-β levels in epithelial conditioned medium were up to 5 times greater than that in keratocyte conditioned medium. • Conclusions: The results indicate that corneal cells maintain tissue homeostasis and modulate the wound healing response via paracrine/autocrine pathways. Received: 6 February 1997 Revised version received: 2 April 1997 Accepted: 10 April 1997     

The effects of growth factors and conditioned media on the proliferation of human corneal epithelial cells and keratocytes

• Background: As growth factors play an important role in epithelial wound repair, we evaluated the effect of exogenous growth factors in the presence and absence of corneal epithelial and keratocyte conditioned medium on human corneal epithelial cell and keratocyte proliferation. • Methods: Preconfluent cultures of human corneal epithelial cells or stromal keratocytes were exposed to varying concentrations of EGF, TGF-β or bFGF in the presence or absence of human corneal epithelial or stromal keratocyte conditioned medium. Cell numbers were determined after 48 h incubation. RIA and ELISA were used to quantify the levels of EGF, TGF-β and bFGF in conditioned media. • Results: EGF and bFGF increased, while TGF-β decreased, the proliferation of both cell types in a dosedependent manner. Epithelial cell conditioned medium inhibited, and keratocyte conditioned medium stimulated, the proliferation of both cell types. The proliferative effects of EGF, TGF-β and bFGF in the presence of keratocyte conditioned medium were additive for both cell types. By contrast, the addition of exogenous growth factors was unable to overcome the inhibitory potential of epithelial conditioned medium. Both conditioned media contained significant levels of bFGF, but TGF-β levels in epithelial conditioned medium were up to 5 times greater than that in keratocyte conditioned medium. • Conclusions: The results indicate that corneal cells maintain tissue homeostasis and modulate the wound healing response via paracrine/autocrine pathways.     

Effects of transforming growth factor β on corneal epithelial and stromal cell function in a rat wound healing model after excimer laser keratectomy

· Background: Transforming growth factor β (TGF-β) regulates extracellular matrix deposition, cell proliferation, and migration, and is expressed in cornea. TGF-β is thought to be involved in the corneal wound healing process. · Methods: The central corneal area (3 mm in diameter) of Lewis rats was ablated using PTK mode excimer laser and the wound healing process was observed at 12 and 24 h and 2, 5, 10, and 30 days after treatment. The expression of TGF-β1, -β2 and -β3, TGF-β type I and type II receptors, α3, α5, β4 integrin subunits, laminin and fibronectin was studied immunohistochemically. Antibody neutralizing TGF-β1, -β2 and -β3 was administered intraperitoneally, 50 μg daily, for 5 days after the laser treatment to investigate the effects of TGF-β function blockade. · Results: At the leading edge of the regenerating epithelium, no TGF-β type I and type II receptors and β4 integrin subunits were expressed after 24 h. Regenerating epithelium covered the ablated area after 2 days. An abnormal fibrotic layer was formed in the subepithelial area. This layer contained round-shaped cells in the stroma in the early stage (2–5 days after laser ablation) and spindle-shaped fibroblast-like keratocytes after 10 days. Laminin and fibronectin expression increased in the fibrotic layer. The increased stromal cells expressed TGF-β isoforms and TGF-β receptors. Neutralizing TGF-β inhibited the stromal cell increase in the laser ablated area after 5 days. · Conclusion: TGF-β may be involved in epithelial cell migration and stromal cell reaction during the corneal wound healing process after excimer laser ablation in rat models. Received: 24 June 1997 Revised version received: 5 January 1998 Accepted: 5 February 1998     

兔视网膜光凝术后组织形态学改变的动态观察

目的:研究532nm激光TsoⅢ级光斑光凝色素家兔视网膜后光凝斑组织形态改变的病理特征。方法:(1)18只色素家兔按光凝后观察时间1,3,7,14,21d及28d分为6组,每组3只。每只(双眼)用532nm激光TsoⅢ级光斑光凝色素家兔视网膜后极部30点(532nm,450mW,100μm,0.05s)。(2)光凝后不同观察时间点行荧光素眼底血管造影(fluorescence fundus angiography,FFA)观察光凝斑FFA特点;取光凝区眼球壁制作标本,进行光镜和透射电镜观察并定量分析视网膜光凝斑大小、视网膜细胞存活率的动态变化。结果:(1)荧光血管造影:光凝后3d,部分光凝斑为高荧光,荧光渗漏随时间延长逐步减轻,至光凝后14d,荧光渗漏基本消失。(2)组织病理学检查:光凝斑区域视网膜各层组织细胞结构破坏;光凝斑周围光感受器细胞的凋亡或坏死和神经节细胞的损伤;继后色素上皮细胞、M櫣ller细胞和成纤维细胞增生修复破坏区。(3)形态学定量指标:光凝后第1d视网膜光凝斑直径最大为116.4±9.6μm,比参照光斑(75μm)增大了55.2%,随后有下降的趋势,21d后为82.8±5.4μm趋于稳定。视网膜细胞存活率光凝后第1d最小为(29.5±4.2)%,随后有上升的趋势,21d为(48.2±4.4)%趋于稳定。结论:视网膜光凝TsoⅢ级光斑会导致光凝斑周围视网膜感觉神经细胞的非选择性、扩展性损伤。     

Effects of local administration of interferon-beta on proliferation of the retinal pigment epithelium]

PURPOSE: We demonstrated effects of local administration of human interferon (IFN)-beta on the repair process of the rabbit retinal pigment epithelium (RPE). MATERIAL AND METHODS: We used adult pigmented rabbits in this experiment. We measured IFN-beta levels in the ocular tissues after sub-Tenon administration of human IFN-beta by means of enzyme-linked immunosorbent assay (ELISA) methods. Laser photocoagulation in moderate intensity was applied after IFN administration. The repair process of the RPE in laser lesion sites was examined histopathologically. RESULTS: Locally administrated IFN spread by diffusion into the intraocular tissues. The highest IFN level was detected in the choroid. In eyes treated with IFN, RPE cells proliferated vigorously to the center of the photocoagulated lesions on early stages after laser photocoagulation. Proliferation of RPE cells after laser photocoagulation was remarkable in eyes treated with large amounts of IFN. CONCLUSION: It was demonstrated histopathologically that sub-Tenon administration of IFN-beta promoted proliferation of RPE cells during the repair process after laser photocoagulation.     

Therapeutic range of repetitive nanosecond laser exposures in selective RPE photocoagulation

• Background: The aim of this study was to investigate whether selective damage the RPE while sparing the adjacent photoreceptors is possible with repetitive 200-ns pulses of Nd:YAG laser (532 nm) and what potential side effects can be expected with higher pulse energies. • Methods: We irradiated the retinas of 19 eyes of 10 chinchilla rabbits with 500 pulses from a Nd:YAG laser, each 200 ns in duration, at a repetition rate of 500 Hz (158 μm, 0–120 μJ). Threshold curves for different effects were established. Representative lesions were investigated by light and transmission electron microscopy. • Results: It was possible to produce lesions, which were only visible by fluorescein angiography. The ED₅₀ threshold energy per pulse for visibility by fluorescein angiography was 2.1 μJ per pulse, for visibility by ophthalmoscopy 8.6 μJ. Bubble formation, an uncommon phenomenon in retinal photocoagulation, occurred at energies of 15–25 μJ. Hemorrhage occurred at surprisingly high energy levels of more than 100 μJ. Histology performed on lesions visible only by angiography showed damage primarily to the RPE and outer segments, with very little damage to some inner segments dependent on the energy used. • Conclusions: Selective RPE damage is possible with repetitive 200-ns laser pulses and appropriate energy; however, the collateral damage to the adjacent retina is more pronounced than with repetitive microsecond laser pulses. There is no risk of hemorrhage of retinal photocoagulation with the repetitive 200-ns laser pulses at low energy levels which would be used clinically. Received: 17 January 1997 Received version received: 10 July 1997 Accepted: 31 July 1997     

Immunolocalization of TGF-β1, -β2 and -β3, and TGF-β receptors in human lens capsules with lens implants

Purpose: To establish the alteration in expression pattern of transforming growth factor (TGF)-βs and their receptors during repair of lens capsules after cataract surgery, we immunohistochemically located TGF-β isoforms and their receptors in human lens capsules before and after cataract surgery.  Methods: Ten post-cataract surgery capsular specimens were obtained during vitrectomy. Three sections of the anterior capsules were obtained during cataract surgery. A whole lens capsular bag immediately after lens extraction was obtained during vitrectomy. Cryosections of these specimens were processed for immunohistochemical analysis for TGF-β1, TGF-β2, TGF-β3, TGF-β receptor type I (TβR-I), type II (TβR-II) and type III (TβR-III), and were observed under light micros-copy. Results: Lens epithelial cells (LECs) lining the inner surface of the anterior capsules exhibited immunoreactivity for TGF-β2 and TβR-II. Immunoreactivity for TGF-β1, -β3, TβR-I and TβR-III was negative. In the whole capsular bag specimen, equatorial LECs were positive for TGF-β1 and -β2, but not for -β3. In post-cataract surgery specimens, antibodies for each TGF-β isoform labelled either the LECs or ECM accumulated on the capsules. Post-surgical LECs expressed TβR-I and TβR-II, and had also TβR-III in seven of the nine specimens examined.  Conclusion: Expression pattern of TGF-β s in quiescent LECs showed regional heterogeneity. Anterior LECs exhibited TGF-β2 immunoreactivity, while equatorial LECs were positive for TGF-β1 and -β2. Quiescent LECs expressed TβR-II. LECs proliferating around IOLs expressed proteins of each TGF-β isoform and each TβR. TGF-β s were also localized in the ECM on capsules undergoing repair. TGF-β3, TβR-I and TβR-III are up-regulated in LECs after cataract surgery. Received: 6 May 1999 Revised: 4 August 1999 Accepted: 30 August 1999