August 1999
Volume 40, Issue 9
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Glaucoma  |   August 1999
Transforming Growth Factor-β1, -β2, and -β3 In Vivo: Effects on Normal and Mitomycin C–Modulated Conjunctival Scarring
Author Affiliations
  • M. Francesca Cordeiro
    From the Wound Healing Research and Glaucoma Units, Department of Pathology, Moorfields Eye Hospital and Institute of Ophthalmology; the
  • Martin B. Reichel
    Department of Molecular Genetics, Institute of Ophthalmology, London, United Kingdom; and the
  • Jennifer A. Gay
    From the Wound Healing Research and Glaucoma Units, Department of Pathology, Moorfields Eye Hospital and Institute of Ophthalmology; the
  • Fabiana D’Esposita
    Department of Molecular Genetics, Institute of Ophthalmology, London, United Kingdom; and the
  • Robert A. Alexander
    From the Wound Healing Research and Glaucoma Units, Department of Pathology, Moorfields Eye Hospital and Institute of Ophthalmology; the
  • Peng T. Khaw
    From the Wound Healing Research and Glaucoma Units, Department of Pathology, Moorfields Eye Hospital and Institute of Ophthalmology; the
Investigative Ophthalmology & Visual Science August 1999, Vol.40, 1975-1982. doi:
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      M. Francesca Cordeiro, Martin B. Reichel, Jennifer A. Gay, Fabiana D’Esposita, Robert A. Alexander, Peng T. Khaw; Transforming Growth Factor-β1, -β2, and -β3 In Vivo: Effects on Normal and Mitomycin C–Modulated Conjunctival Scarring. Invest. Ophthalmol. Vis. Sci. 1999;40(9):1975-1982.

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

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Abstract

purpose. To compare the effects of the three human isoforms of transforming growth factor (TGF)-β in vivo using a mouse model of conjunctival scarring, both in normal eyes and after treatment with MMC, with a view to delineating the role of this growth factor in glaucoma filtration surgery.

methods. Application of recombinant human TGF-β was assessed in a prospective, randomized study of mouse conjunctival scarring, in which subconjunctival TGF-β1, -β2, and -β3 (all 10−9 M) were compared with control (phosphate-buffered saline [PBS] carrier) and mitomycin C (MMC; 0.4 mg/ml) treatment at 6 hours, and 1, 3, and 7 days after surgery (six eyes/treatment/time point). Effects of TGF-β2 on eyes previously treated with MMC were also assessed. Histologic studies of enucleated eyes were performed to analyze development of the scarring response, extracellular matrix deposition, and the inflammatory cell profile.

results. All three isoforms of TGF-β behaved in a similar manner in vivo, being associated with a rapid-onset and exaggerated scarring response compared with control and MMC treatment. TGF-β–treated eyes showed evidence of an earlier peak in inflammatory cell activity (P < 0.05) and increased collagen type III deposition (P < 0.05). TGF-β2 treatment significantly stimulated scarring after MMC application (P < 0.05).

conclusions. TGF-β1, -β2, and -β3 appear to have similar actions in vivo and stimulate the conjunctival scarring response. Application of TGF-β2 modified the effects of MMC. All TGF-β isoforms may be potent modulators of the conjunctival scarring response. These studies indicate that TGF-β2 may naturally modify the antiscarring effects of antimetabolites such as MMC in glaucoma filtration surgery.

The conjunctival scarring response is a major determinant of morbidity and visual prognosis in a wide spectrum of ocular diseases including cicatricial conditions (e.g., trachoma and pemphigoid) and those in which the results of treatment depend on the healing response after surgery, such as glaucoma. The commonest cause of failure of glaucoma filtration surgery is the occurrence of subconjunctival scarring at the bleb and sclerostomy sites, 1 2 with increased scar deposition being associated with poor control of postoperative intraocular pressure. 
Although the role of growth factors in conjunctival scarring is believed to be important, that of transforming growth factor-β (TGF-β), the most potent growth factor involved in wound healing throughout the body, 3 4 5 6 is unclear. There are three isoforms of TGF-β found in humans. Of these, TGF-β1 and -β2 are known to stimulate greatly the dermal scarring response. 4 5 6 7 8 9 10 The actions of the third isoform, TGF-β3, in wound healing are less well established, with some studies suggesting that it may actually inhibit in vivo scarring. 4 11  
Although the effects of exogenous TGF-β have been studied in skin, the actions of all three isoforms after administration in the eye have not been examined to date. However, it is known that compared with TGF-β1 and -β3, TGF-β2 is the predominantly expressed ocular isoform, having been identified in normal and diseased eyes 12 13 and implicated in the pathogenesis of several ocular scarring diseases such as proliferative vitreoretinopathy and cataract formation. 14 15 More recent work, however, has suggested TGF-β1 and -β3 may be important in the fibrosis occurring in the cicatrizing disease, ocular pemphigoid. 16  
The conjunctival scarring response in glaucoma filtration surgery is thought to be greatly influenced by the passage of aqueous through the surgical site, and in particular, growth factors in the aqueous such as TGF-β. 17 However, compared with the other growth factors found in aqueous humor, TGF-β2 has been shown to be the most potent 18 and is significantly raised in glaucomatous eyes. 19 In the light of these findings, our study specifically investigates the effects of TGF-β on the conjunctival scarring response. 
Using our recently established model of conjunctival scarring in the mouse eye, 20 we have compared the effects of all three TGF-β isoforms. The model is unique because unlike fistulizing glaucoma surgery, which causes a complex, dynamically changing wound environment involving the sclera, the conjunctiva, the aqueous humor, its various cytokines and chemical mediators, it consists of a wound-healing response that is specifically localized to the subconjunctival space. We have investigated the conjunctival scarring response induced by TGF-β in the mouse eye, using histologic techniques that identify collagen, elastic-related fibers, and inflammatory cells and have compared this to control and the commonly used modulating agent mitomycin C (MMC). In addition, we have assessed the effects of TGF-β2 on MMC-treated eyes. 
Methods
Materials
The exogenous TGF-βs used in these studies were all recombinant human proteins. TGF-β2 and -β3 were generous gifts of Ciba Geigy, Switzerland, and TGF-β1 was a gift from Professor Gregory Schultz, University of Florida, Gainesville. All TGF-β injections were reconstituted from stock solutions of the active form (1 mg/ml) in phosphate buffered saline (PBS) containing 1% bovine serum albumin (Sigma) at a concentration of 10−9 M. PBS was used as the carrier and the control. Mitomycin-C (MMC, Kyowa, Essex, United Kingdom) was used at the clinically used concentration of 0.4 mg/ml. 
Animals
All experiments were performed on 6-week-old BALB/c mice, and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice eyes were randomly allocated to one of seven treatments (six eyes/treatment/time point): subconjunctival injections of a single of 25 μl of TGF-β1, -β2, or -β3 (10−9 M), or control (PBS carrier); MMC (0.4 mg/ml); or dual therapy consisting of subconjunctival injections of MMC (0.4 mg/ml) followed 24 hours later by either TGF-β2 (10−9 M) (MMC+TGF-β2), or PBS control (MMC+control). 
Surgery was performed using the method previously described. 20 Briefly, all animals underwent general anesthesia with intraperitoneal injections (0.2 ml Hypnorm; Janssen Pharmaceutical, Oxford, UK; and Hypnovel; Roche, Welwyn Garden City; UK) mixed 1:1:6 with sterile distilled water. A subconjunctival injection of 25 μl of each test substance with a 27-gauge needle was given at the same location in each animal, under visualization with a microscope (OPMI MDU; Carl Zeiss, Oberkochen, Germany). This position was marked with a corneal 10-0 nylon suture at postmortem but before enucleation. Mice were assessed clinically and killed by cervical dislocation at 6 hours and 1, 3, and 7 days after surgery. 
Histochemistry
All enucleated eyes were prepared for histologic analysis after fixation for 24 hours with 10% buffered formaldehyde and embedded whole in paraffin wax. Development of scar tissue was studied in sequential 5-μm thick sections using the following special stains: hematoxylin and eosin to assess cellularity, van Gieson and picrocirius red (viewed under polarized light) to demonstrate collagen deposition, and aldehyde fuchsin for elastic and elaunin fibers together with oxidation-aldehyde fuchsin for additionally demonstrating oxytalan fibers. In addition, immunofluorescence was used to demonstrate collagen types I and III. Primary antibodies were goat anti-mouse collagen I (1:25 dilution, Europath, Southern Biotechnology) and chick anti-mouse collagen III antibodies (1:25 dilution, Europath, Southern Biotechnology) with a secondary fluorescein isothiocyanate (FITC)-labeled chick anti-goat and rabbit anti-chick IgG antibodies (1:100 dilution, Serotec). Photographs were taken at an excitation–emission wavelength of 490 and 525nm. 
Lymphocyte and macrophage identification was performed using the lymphocyte CD3 rat anti-human antibody (1:50 dilution, Serotec) and macrophage F4/80 rat anti-mouse antibody (1:20 dilution, Serotec) as primary antibodies incubated for 2 hours, followed by a further 2-hour incubation with a FITC-labeled goat anti-rat IgG secondary antibody (1:100 dilution, Serotec). Fibroblasts were identified using phalloidin, which stains the fibroblast cytoskeleton. 21 Briefly, sections were soaked in Tris-buffered saline (TBS; 25 mM Tris, 140 mM NaC1, 3 mM KCl [pH 7.4]) before a 2-hour incubation in FITC-phalloidin (Sigma) at 2.5 μg/ml in TBS. Sections were then rinsed and mounted in PBS and viewed by fluorescence microscopy. 
Statistical Analysis
Sequential sections were assessed for cellularity profile and extracellular matrix deposition by three independent and masked observers (MFC, RA, JG), using the grading system modified from Shah et al. 22 (scale: 0–4; where 0 is normal conjunctiva; 1 is 1–25% of normal; 2 is 2–50% of normal; 3 is 51–75% of normal; and 4 is >75%; no prefix, more than; − prefix, less than). Parameters assessed included total cellularity; collagen types I and III and elastic fiber deposition; and fibroblast, macrophage, and lymphocyte profiles. For each treatment group a mean grade per parameter at each time point (with 95% confidence intervals [CI]) was calculated. 
Analysis was performed using computer software (SPSS for Windows; SPSS; Chicago, IL) at individual time points using a one-way analysis of variance. All treatments were compared with control (PBS). The observed significance levels from multiple comparisons were adjusted using the Bonferroni test, with P < 0.05 indicating significance. 
Results
Effects of TGF-β Isoforms
Mouse eyes showed evidence of a visible subconjunctival bleb after all subconjunctival injection, which was macroscopically apparent until 24 hours (Fig. 1) . Little difference between macroscopic appearances of injected eyes was noted thereafter, and no clinical evidence of periocular inflammation was seen at any time after surgery. 
Histologic evaluation showed a characteristic response to subconjunctival administration of TGF-β (Fig. 2) . Semiquantitative analysis revealed all TGF-β isoforms produced a similar scarring response. There was no significant difference in any of the parameters studied between TGF-β isoforms at any time point including total cellularity (Fig. 3 A). 
Compared with control, TGF-β treatment was associated with an earlier peak in the appearance of macrophages and lymphocytes (Figs. 3B 3C ; P < 0.05). Maximal numbers of macrophages and lymphocytes for TGF-β treatment occurred at 6 hours, compared with control in which maximal counts occurred at day 1. No significant difference between treatment groups was seen in fibroblast activity, although TGF-β was associated with increased fibroblast activity with time. 
TGF-β2 significantly stimulated collagen III deposition compared with control at day 7 (Fig. 3E ; P < 0.05). Although both TGF-β1 and -β3 also showed increased collagen III deposition compared with control, this did not reach statistical significance. No statistically significant difference in collagen I, picrocirius and aldehyde fuchsin staining was associated with TGF-β treatment, although there was a trend toward more picrocirius staining with TGF-β compared with control after day 1. 
Effect of MMC
As expected, there was a significant reduction in total cellularity associated with MMC treatment compared with control and all TGF-β-treated eyes (days 3 and 7; P < 0.05). In comparison with control, MMC treatment significantly inhibited macrophage and lymphocyte levels on day 1 and fibroblast activity on days 1, 3, and 7 (P < 0.05). Macrophage activity was significantly reduced by MMC treatment compared with TGF-β1, -β2, and -β3 at 6 hours (P < 0.05), and there was a significant difference in lymphocyte and fibroblast levels in MMC and TGF-β2-treated eyes at 6 hours and 7 days, respectively (P < 0.05). 
A reduction in extracellular matrix components was also associated with MMC treatment. Compared with control, this was significant on days 3 and 7 for collagen III (P < 0.05), day 7 for collagen I (P < 0.05), day 3 for elastic fiber, and day 7 for picrocirius red staining (P < 0.05). Differences among MMC and TGF-β1, -β2, and -β3 treatments were present with a reduction in collagen III on days 3 and 7 (P < 0.05), elastic fibers at 6 hours and on day 3 (P < 0.05), and picrocirius red staining on day 7. 
Assessment of Modulation of MMC Effects by TGF-β2
Figure 4 shows histologic sections characteristics of the conjunctival scarring response after treatment with MMC. Figures 4C and 4F demonstrate the modulation of the antiscarring effects associated with MMC by a subsequent injection of TGF-β2 (10−9 M), 7 days after treatment. Also of note is the appearance of abnormal conjunctival epithelial cells in all MMC-treated eyes, as shown in Figures 4A 4B and 4C
Comparisons of histologic effects of MMC followed by PBS (MMC+control) and MMC followed by TGF-β2 (MMC+TGF-β2) treatment are shown in Figure 5 . These results should also be compared with single applications of TGF-β2 injections, displayed in Figure 3
TGF-β2 treatment (MMC+TGF-β2) was associated with significantly more cellular activity on day 3 (Fig. 5A ; P < 0.05), compared with either MMC+control or MMC treatment alone. TGF-β2 significantly stimulated levels of macrophages on days 1 and 7 (Fig. 5B ; P < 0.05), and lymphocytes and fibroblasts on days 3 and 7 (Fig. 5C 5D ; P < 0.05). Although MMC+TGF-β2 was associated with greatest cellular activity, both groups showed peak macrophage, lymphocyte, and fibroblast activity at 6 hours. 
TGF-β2 treatment was associated with stimulation of extracellular matrix components. Compared with MMC+control, this was significant on days 3 and 7 for collagen III and collagen I (Figs. 5E 5F ; P < 0.05). Differences in picrocirius red staining was also present on days 1, 3, and 7(Fig. 5D ; P < 0.05). Although not significant, there was a trend for TGF-β2 treatment to be associated with greater levels of all studied extracellular matrix components than in control (Fig. 5D)
Discussion
Our studies have shown that all three human TGF-β isoforms, when applied exogenously and at the same concentration, produced a similar conjunctival scarring response. This response was characterized by an earlier and more pronounced peak in inflammatory cell activity with evidence of enhanced fibroblast activity in TGF-β treatment groups compared with control. In addition, TGF-β2 was associated with increased collagen III deposition, although no significant difference between TGF-β and control groups was demonstrated in extracellular matrix architecture. 
Little is known about the effect of exogenous TGF-β in conjunctival scarring. However, elsewhere in the eye, TGF-β has been advocated as a biologic chorioretinal “glue” for use in repairing retinal tears 23 and macular holes. 24 Glaser et al. 24 suggested that visual acuity after TGF-β2 treatment in macular hole surgery significantly improved in a dose-related manner (range, 0.28–5.32 × 10−7 M TGF-β2). 
Early work by Roberts et al. 10 showed that a subcutaneous injection of TGF-β1 and -β2 (0–16 × 10−7 M) in newborn mice, stimulated the formation of granulation tissue, associated with induction of angiogenesis, increased fibroblast number, and collagen deposition. TGF-β administration into the peritoneum has also been shown to induce fibrosis. 25 Williams et al. 25 demonstrated that application of 16 × 10−8 M TGF-β1 after surgical injury to the uterine horns in rats, significantly increased the number of adhesions formed after surgery, with evidence of an increased number of inflammatory cells and fibroblasts, histologically. 25  
Application of TGF-β2 (4 and 40 × 10−8 M) on healing fractures in the rabbit, demonstrated that at the higher dose, TGF-β2 promoted callus formation in stable conditions. 26 However, in unstable conditions, TGF-β2 was found to be opposite in that they retarded and reduced bone and cartilage formation in the callus. TGF-β2 was not found to accelerate fracture healing. This is suggestive that the actions of TGF-β2 are determined by its extracellular environment. 
The effects of exogenous TGF-β application have also been studied in several models of dermal wound healing. Shah et al. 22 have demonstrated that exogenous application of TGF-β1 to a linear incisional wound in rat skin affects the response in a dose-dependent manner (range, 0.8–20 × 10−9 M). At 4 and 20 × 10−9 M concentrations of TGF-β1, wounds showed increased vascularity and extracellular matrix deposition compared with controls, with evidence of scarring. However, no significant difference in cellularity was noted between TGF-β1 and control groups. 
In one of the few studies comparing the in vivo effects of all three TGF-β isoforms, Shah et al. 4 applied exogenous TGF-β1, -β2, and -β3 to the same incisional rat model of dermal scarring described above. All isoforms were assessed at different concentrations of 0.04, 0.4, 4, and 20 × 10−9 M. Differences between the isoforms were noted at 4 and 20 × 10−9 M in increased fibronectin in association with TGF-β1 and -β2 only, increased collagen fibril organization with TGF-β3 only, decreased monocyte and macrophage profile of wounds treated with TGF-β3 (compared with TGF-β1 and -β2 which were similar to control), and increased collagen I and III in TGF-β1 and -β2 treatments only. All isoforms were associated with increased vascularity and angiogenesis. The authors suggest that TGF-β3 inhibited scarring and promoted better collagen organization, compared with TGF-β1 and -β2, which stimulated dermal scarring. 
However, Cox 11 has shown that TGF-β3 application, both in thermal wounds in mice and incisional and second-intention wounds in rats, stimulates the cutaneous scarring response. This stimulation by TGF-β3, similar to that associated with TGF-β1 and -β2, resulted in accelerated re-epithelialization, increased fibroblast and phagocyte activity, and increased protein, DNA, and collagen production. 
Our results, similar to those of Cox, 11 suggest that in conjunctival scarring all three TGF-β isoforms have similar stimulatory effects. This is supported by our data from in vitro work in our laboratory investigating effects of TGF-β on conjunctival fibroblasts. 27 The TGF-β used in this study was 100% active. This is different from cellular TGF-β, which, when produced, is secreted in a latent form that has to be activated into a mature, active form. It is known that only between 22% and 61% of TGF-β found in aqueous is in its active rather than its latent form 17 28 and the average concentration of active TGF-β2 in normal aqueous is between 0.73 and 10.98 × 10−11 M compared with 10−9 M, a much higher concentration, used in our study. 
Using the mouse model of conjunctival scarring, we have shown that TGF-β2 applied to mice conjunctiva after MMC treatment modulated and significantly reversed the antiscarring effects of MMC. Specifically, this was demonstrated by TGF-β2 treatment stimulating cellular activity including macrophages, lymphocytes, and fibroblasts compared with either control or MMC treatment alone. In addition, TGF-β2 treatment was associated with increased collagen type I and III deposition. 
The use of TGF-β2 as a modulating agent to counteract effects of MMC treatment, has been shown by other investigators. 29 30 Doyle et al. 30 demonstrated that a single peribleb injection of 4 × 10−7 M TGF-β2 effectively treated 50% of bleb leaks deliberately induced in a rabbit model of MMC-assisted filtration surgery. Histologic examination revealed increased peribleb cellularity and denser collagen deposition in TGF-β eyes compared with control eyes. 
The present mouse study also shows the possible use of TGF-β2 as a modulator of the potent antiscarring effects of conjunctival MMC. This is important because although MMC has been shown to be highly effective as an adjuvant treatment for preventing postoperative scarring in glaucoma filtration surgery, its use is associated with several complications. These include the production of thin, avascular cystic blebs with the attendant risks of persistent hypotony, bleb leaks, and endophthalmitis. TGF-β2 may offer another therapeutic strategy for enhancing healing after MMC use, although the method of its instillation would be very important. Our studies on MMC bleb sizes 31 suggest that a peribleb injection (as administered by Doyle et al. 30 ) would not be suitable, because leaking blebs are often thin-walled cystic blebs, with an increase in cellularity around the bleb itself. A peribleb injection of TGF-β2 would further increase the degree of peribleb cellular activity contributing to an increase in surrounding scar tissue fibrosis and contraction, which in turn would produce even smaller blebs with thinner walls. It would seem more appropriate that TGF-β2 be applied either transconjunctivally or with an intrableb injection. Obviously, these methods need further investigation. 
In summary, our results show that all three TGF-β human isoforms have similar actions in subconjunctival scarring in vivo. They each stimulate the scarring response in an exaggerated and rapid manner compared with control and MMC. Application of TGF-β2 modulated the antiscarring effects of MMC. This may be an important clinical finding and suggests that TGF-β is a potent modulator of the conjunctival scarring response and may naturally modify the antiscarring effects of antimetabolites such as MMC in glaucoma filtration surgery. 
 
Figure 1.
 
Subconjunctival injections of 25 μl of test article were administered 0.5 mm behind the limbus and in proximity with the posterior canthal angle of each mouse eye. A subconjunctival bleb was visible in all eyes after the subconjunctival injection and was macroscopically apparent until 24 hours.
Figure 1.
 
Subconjunctival injections of 25 μl of test article were administered 0.5 mm behind the limbus and in proximity with the posterior canthal angle of each mouse eye. A subconjunctival bleb was visible in all eyes after the subconjunctival injection and was macroscopically apparent until 24 hours.
Figure 2.
 
Histologic demonstration of effect of TGF-β2 on the conjunctival scarring response. Photographs (A through D) show hematoxylin and eosin stains of subconjunctival bleb area treated with control (PBS) or TGF-β2 (10−9) at day 1 (A, B showing increased cellularity) and day 7 (C, D), respectively. Aldehyde fuchsin staining reveals elastic fibers (dark blue fibrillar stain) shown in (E, F, G), with a suggestion of early deposition with TGF-β treatment (F). Collagen architecture is demonstrated with picrocirius red (I through L) and collagen type III immunofluorescence (M through P) and illustrates increased collagen deposition with TGF-β treatment compared with control (L and P). Magnification, ×25.
Figure 2.
 
Histologic demonstration of effect of TGF-β2 on the conjunctival scarring response. Photographs (A through D) show hematoxylin and eosin stains of subconjunctival bleb area treated with control (PBS) or TGF-β2 (10−9) at day 1 (A, B showing increased cellularity) and day 7 (C, D), respectively. Aldehyde fuchsin staining reveals elastic fibers (dark blue fibrillar stain) shown in (E, F, G), with a suggestion of early deposition with TGF-β treatment (F). Collagen architecture is demonstrated with picrocirius red (I through L) and collagen type III immunofluorescence (M through P) and illustrates increased collagen deposition with TGF-β treatment compared with control (L and P). Magnification, ×25.
Figure 3.
 
Charts (A through D) show the effects of exogenous TGF-β on the conjunctival scarring response. All TGF-β isoforms behave similarly, including increasing total cellularity in the subconjunctival bleb area (A). No significant difference was noted between isoforms and control. Analysis of the cellular profile revealed TGF-β treatment to be associated with increased macrophage and lymphocyte activity earlier than control (B, C; day 1). Collagen III deposition was stimulated significantly more by TGF-β on day 7 (E). No other statistically significant difference in extracellular matrix components was noted between TGF-β treatment groups and control. Error bars, 95% CI. *Activity of TGF-β2 significantly different from control (P < 0.05).
Figure 3.
 
Charts (A through D) show the effects of exogenous TGF-β on the conjunctival scarring response. All TGF-β isoforms behave similarly, including increasing total cellularity in the subconjunctival bleb area (A). No significant difference was noted between isoforms and control. Analysis of the cellular profile revealed TGF-β treatment to be associated with increased macrophage and lymphocyte activity earlier than control (B, C; day 1). Collagen III deposition was stimulated significantly more by TGF-β on day 7 (E). No other statistically significant difference in extracellular matrix components was noted between TGF-β treatment groups and control. Error bars, 95% CI. *Activity of TGF-β2 significantly different from control (P < 0.05).
Figure 4.
 
Histologic assessment at day 7 of mice eyes treated with subconjunctival injections of MMC (0.4 mg/ml) followed by no treatment, PBS, or TGF-β2 (10−9 M) injections 24 hours later. Hematoxylin and eosin histologic staining (A, B, C) show the hypocellular appearance of the subconjunctival bleb area with MMC (A, B) and the increased cellularity associated with TGF-β2 treatment (C). Picrocirius red stain demonstrating collagen architecture (D, E, F) reveals little extracellular matrix in the MMC-treated areas (D, E). Exogenous TGF-β2 appears to stimulate extracellular matrix deposition (F) and effectively reverse the antiscarring effects of MMC. Magnification ×40.
Figure 4.
 
Histologic assessment at day 7 of mice eyes treated with subconjunctival injections of MMC (0.4 mg/ml) followed by no treatment, PBS, or TGF-β2 (10−9 M) injections 24 hours later. Hematoxylin and eosin histologic staining (A, B, C) show the hypocellular appearance of the subconjunctival bleb area with MMC (A, B) and the increased cellularity associated with TGF-β2 treatment (C). Picrocirius red stain demonstrating collagen architecture (D, E, F) reveals little extracellular matrix in the MMC-treated areas (D, E). Exogenous TGF-β2 appears to stimulate extracellular matrix deposition (F) and effectively reverse the antiscarring effects of MMC. Magnification ×40.
Figure 5.
 
Charts (A through H) show the effects of exogenous TGF-β2 on conjunctival scarring response after treatment with MMC (0.4 mg/ml; MMC+TGF-β2). Comparison between MMC+TGF-β2 treatment and MMC+control (MMC followed by PBS) in total cellularity in the subconjunctival bleb area is shown in (A). Cellular profile analysis showed a significant increase in macrophages on days 1 and 7 and lymphocytes and fibroblasts on days 3 and 7 after TGF-β2 treatment (B, C, D). Differences were also associated with TGF-β treatment in extracellular matrix deposition: Collagens type I and III were significantly stimulated by TGF-β on days 3 and 7 (E, F). Picrocirius staining was also significantly increased on days 1, 3, and 7 (G) after TGF-β2. No significant difference in other extracellular matrix components was found. *Activity of TGF-β2 significantly different from control (P < 0.05). Error bars, 95% CI.
Figure 5.
 
Charts (A through H) show the effects of exogenous TGF-β2 on conjunctival scarring response after treatment with MMC (0.4 mg/ml; MMC+TGF-β2). Comparison between MMC+TGF-β2 treatment and MMC+control (MMC followed by PBS) in total cellularity in the subconjunctival bleb area is shown in (A). Cellular profile analysis showed a significant increase in macrophages on days 1 and 7 and lymphocytes and fibroblasts on days 3 and 7 after TGF-β2 treatment (B, C, D). Differences were also associated with TGF-β treatment in extracellular matrix deposition: Collagens type I and III were significantly stimulated by TGF-β on days 3 and 7 (E, F). Picrocirius staining was also significantly increased on days 1, 3, and 7 (G) after TGF-β2. No significant difference in other extracellular matrix components was found. *Activity of TGF-β2 significantly different from control (P < 0.05). Error bars, 95% CI.
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Figure 1.
 
Subconjunctival injections of 25 μl of test article were administered 0.5 mm behind the limbus and in proximity with the posterior canthal angle of each mouse eye. A subconjunctival bleb was visible in all eyes after the subconjunctival injection and was macroscopically apparent until 24 hours.
Figure 1.
 
Subconjunctival injections of 25 μl of test article were administered 0.5 mm behind the limbus and in proximity with the posterior canthal angle of each mouse eye. A subconjunctival bleb was visible in all eyes after the subconjunctival injection and was macroscopically apparent until 24 hours.
Figure 2.
 
Histologic demonstration of effect of TGF-β2 on the conjunctival scarring response. Photographs (A through D) show hematoxylin and eosin stains of subconjunctival bleb area treated with control (PBS) or TGF-β2 (10−9) at day 1 (A, B showing increased cellularity) and day 7 (C, D), respectively. Aldehyde fuchsin staining reveals elastic fibers (dark blue fibrillar stain) shown in (E, F, G), with a suggestion of early deposition with TGF-β treatment (F). Collagen architecture is demonstrated with picrocirius red (I through L) and collagen type III immunofluorescence (M through P) and illustrates increased collagen deposition with TGF-β treatment compared with control (L and P). Magnification, ×25.
Figure 2.
 
Histologic demonstration of effect of TGF-β2 on the conjunctival scarring response. Photographs (A through D) show hematoxylin and eosin stains of subconjunctival bleb area treated with control (PBS) or TGF-β2 (10−9) at day 1 (A, B showing increased cellularity) and day 7 (C, D), respectively. Aldehyde fuchsin staining reveals elastic fibers (dark blue fibrillar stain) shown in (E, F, G), with a suggestion of early deposition with TGF-β treatment (F). Collagen architecture is demonstrated with picrocirius red (I through L) and collagen type III immunofluorescence (M through P) and illustrates increased collagen deposition with TGF-β treatment compared with control (L and P). Magnification, ×25.
Figure 3.
 
Charts (A through D) show the effects of exogenous TGF-β on the conjunctival scarring response. All TGF-β isoforms behave similarly, including increasing total cellularity in the subconjunctival bleb area (A). No significant difference was noted between isoforms and control. Analysis of the cellular profile revealed TGF-β treatment to be associated with increased macrophage and lymphocyte activity earlier than control (B, C; day 1). Collagen III deposition was stimulated significantly more by TGF-β on day 7 (E). No other statistically significant difference in extracellular matrix components was noted between TGF-β treatment groups and control. Error bars, 95% CI. *Activity of TGF-β2 significantly different from control (P < 0.05).
Figure 3.
 
Charts (A through D) show the effects of exogenous TGF-β on the conjunctival scarring response. All TGF-β isoforms behave similarly, including increasing total cellularity in the subconjunctival bleb area (A). No significant difference was noted between isoforms and control. Analysis of the cellular profile revealed TGF-β treatment to be associated with increased macrophage and lymphocyte activity earlier than control (B, C; day 1). Collagen III deposition was stimulated significantly more by TGF-β on day 7 (E). No other statistically significant difference in extracellular matrix components was noted between TGF-β treatment groups and control. Error bars, 95% CI. *Activity of TGF-β2 significantly different from control (P < 0.05).
Figure 4.
 
Histologic assessment at day 7 of mice eyes treated with subconjunctival injections of MMC (0.4 mg/ml) followed by no treatment, PBS, or TGF-β2 (10−9 M) injections 24 hours later. Hematoxylin and eosin histologic staining (A, B, C) show the hypocellular appearance of the subconjunctival bleb area with MMC (A, B) and the increased cellularity associated with TGF-β2 treatment (C). Picrocirius red stain demonstrating collagen architecture (D, E, F) reveals little extracellular matrix in the MMC-treated areas (D, E). Exogenous TGF-β2 appears to stimulate extracellular matrix deposition (F) and effectively reverse the antiscarring effects of MMC. Magnification ×40.
Figure 4.
 
Histologic assessment at day 7 of mice eyes treated with subconjunctival injections of MMC (0.4 mg/ml) followed by no treatment, PBS, or TGF-β2 (10−9 M) injections 24 hours later. Hematoxylin and eosin histologic staining (A, B, C) show the hypocellular appearance of the subconjunctival bleb area with MMC (A, B) and the increased cellularity associated with TGF-β2 treatment (C). Picrocirius red stain demonstrating collagen architecture (D, E, F) reveals little extracellular matrix in the MMC-treated areas (D, E). Exogenous TGF-β2 appears to stimulate extracellular matrix deposition (F) and effectively reverse the antiscarring effects of MMC. Magnification ×40.
Figure 5.
 
Charts (A through H) show the effects of exogenous TGF-β2 on conjunctival scarring response after treatment with MMC (0.4 mg/ml; MMC+TGF-β2). Comparison between MMC+TGF-β2 treatment and MMC+control (MMC followed by PBS) in total cellularity in the subconjunctival bleb area is shown in (A). Cellular profile analysis showed a significant increase in macrophages on days 1 and 7 and lymphocytes and fibroblasts on days 3 and 7 after TGF-β2 treatment (B, C, D). Differences were also associated with TGF-β treatment in extracellular matrix deposition: Collagens type I and III were significantly stimulated by TGF-β on days 3 and 7 (E, F). Picrocirius staining was also significantly increased on days 1, 3, and 7 (G) after TGF-β2. No significant difference in other extracellular matrix components was found. *Activity of TGF-β2 significantly different from control (P < 0.05). Error bars, 95% CI.
Figure 5.
 
Charts (A through H) show the effects of exogenous TGF-β2 on conjunctival scarring response after treatment with MMC (0.4 mg/ml; MMC+TGF-β2). Comparison between MMC+TGF-β2 treatment and MMC+control (MMC followed by PBS) in total cellularity in the subconjunctival bleb area is shown in (A). Cellular profile analysis showed a significant increase in macrophages on days 1 and 7 and lymphocytes and fibroblasts on days 3 and 7 after TGF-β2 treatment (B, C, D). Differences were also associated with TGF-β treatment in extracellular matrix deposition: Collagens type I and III were significantly stimulated by TGF-β on days 3 and 7 (E, F). Picrocirius staining was also significantly increased on days 1, 3, and 7 (G) after TGF-β2. No significant difference in other extracellular matrix components was found. *Activity of TGF-β2 significantly different from control (P < 0.05). Error bars, 95% CI.
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