Fibronectin is a noncollagenous extracellular matrix glycoprotein that has been shown to be implicated in a variety of cellular properties, particularly those involving the interactions of cells with extracellular structures such as basement membrane or loose connective tissue . Fibronectin has three binding sites, one of which usually binds to cell surface proteins called integrin, the other one to collagen fibers, and one to the proteoglycan . It exists in two main forms termed plasma and cellular fibronectin. The plasma form is synthesized by hepatocytes, endothelial cells, and macrophages and the extracellular form is made by fibroblasts, chondrocytes, endothelial cells, macrophages, as well as certain epithelial cells . Fibronectin is a principal component of the extracellular matrix (ECM), where it is organized into elongated branching fibrils. It is involved in cell-to-cell and cell-to-substrate adhesions, and modulates cellular functions such as cell migration, cytoskeletal organization, differentiation, phagocytosis, wound repair, oncogenic transformation, hemostasis, and embryogenesis . Beside its interaction with components of the cytoskeleton and the ECM, it also interacts with circulating components of the blood clotting and complement systems and with cell surface receptors on a variety of cells including fibroblasts [5,6]. Fibronectin polymerization is a continuous process that plays a pivotal role in determining the mechanical strength of tissues, with nearly 50% of the fibronectin matrix undergoing turnover every 24 h . Fibronectin may increase or decrease in various clinical problems. The reasons for fibronectin loss appear to vary, and include reduced synthesis, reduced binding, and increased rate of degradation. Extensive studies have shown that loss of fibronectin is common, although not universal, concomitant of malignant transformation . In the eye, fibronectin is expressed in a wide variety of tissues in a different manner. It might aid in healing of corneal epithelial defects, influence the metabolism of the trabecular meshwork, and attach the vitreous to the internal limiting membrane of the retina . On the basis of these facts, this study was conducted to evaluate the immunohistochemical expression of fibronectin in the ocular tissues of adult male albino rat.
Materials and methods
This study was carried out in the Histology Department (Faculty of Medicine, Tanta University, Tanta, Egypt) on 10 adult male albino rats, weighing from 180 to 200 gm. They were housed in clean and properly ventilated cages under the same environmental conditions with free access to food and water throughout the whole period of the experiment. Animals were anesthetized and perfused with 4% paraformaldehyde in 0.1 mol/l sodium phosphate buffer (pH7.4) solution. After perfusion, the eyeballs were harvested by careful dissection and were immediately fixed in 4% paraformaldehyde for preparation of paraffin blocks. Five micrometer sections were mounted, dried, and dewaxed in xylene. Rehydrated sections were then incubated with 3% hydrogen peroxide in humidified boxes to block the endogenous activity of peroxidase. Microwave-assisted antigen retrieval was performed for 20 min. Sections were then incubated overnight at 4°C with primary rabbit antirat fibronectin antibody, (NeoMarkers/Lab Vision, Fremont, California, USA) (Mouse monoclonal antibody). After washing with phosphate-buffered solution, sections were incubated with biotinylated goat antirabbit secondary antibody for 30 min and then with streptavidin peroxidase conjugate for 30 min. Sections were then washed with phosphate buffer solution, and incubated with diaminobenzidine chromogen to detect immunoreactivity. Counterstaining was performed by Mayer's hematoxylin. Positive reaction was visualized as brown coloration of the subcellualr and interstitial areas. Negative controls were performed using the same steps, except that phosphate-buffered saline was applied instead of the primary antibody .
Morphometric and statistical analysis
A detailed morphometric analysis of the optical density of fibronectin expression was assessed in the ocular tissues of the different sections from each slide of different animals at a magnification of ×400. This was performed using a Leica Qwin 500 MCO image analysis system (Wetzlar, Germany) at the National Research Center, Cairo, Egypt. Values of the measured parameters were presented as mean±standard deviation. The statistical analysis was processed according to the conventional procedures using the statistical program of social sciences (SPSS) software for windows (version 10.0) (IBM, USA) .
Microscopic analysis showed abolished immunostaining in the negative control specimens (Fig. 1). Immunohistochemical assaying showed wide distribution of fibronectin in all layers of the eyeball of the adult male albino rat. Cornea displayed prominent fibronectin linear staining at the whole thickness of the substantia propria, extending from Bowman's to Descemets' membranes. Bowman's membrane displayed positive-fibronectin reaction, which was indistinguishable from that of the substantia propria. Descemets' membrane displayed mild positive-broadband immunostaining reaction, whereas corneal endothelium did not (Figs 2 and 3). The corneoscleral junction (limbus) displayed staining at the basement membrane of the bulbar conjunctiva, and in the substantia propria of the sclera and around the trabecular spaces of the Fontana (trabecular meshwork) and the canal of schlemm (Fig. 3).
Middle layer revealed fibronectin staining in the stroma of the iris (Figs 3, 4, 6) and ciliary body between the ciliary muscles and around their blood vessels (Figs 3 and 5). The ciliary processes also revealed positive reaction in their core of loose connective tissues (Fig. 5). Fibronectin was expressed in the basement membrane of their epithelium (Figs 4 and 5). The lens displayed fibronectin in the capsule, whereas the epithelium and the lens fibers revealed no reaction (Fig. 6). Choroids displayed positive-fibronectin reaction around large blood vessels of outer choroid and around capillaries of the inner choroid and also mildly in the Bruch's membrane (Fig. 7).
With regard to the retina, moderate immunoreactivity was observed in the outer and inner plexiform layers and a prominent reaction was observed in the internal limiting membrane (Fig. 8).
Morphometry and statistical analysis
Morphometric analysis revealed the differential expression capacity of fibronectin in the different ocular tissues of the eye being prominently presented in the sclera, followed by ciliary body, choroid, iris, cornea, conjunctiva, retina, and finally lens capsule (Table 1 and Histogram 1).
This study investigated fibronectin localization in ocular tissues of adult male albino rat. As demonstrated, corneal fibronectin was expressed abundantly in the corneal stroma and weakly in the Descemet's membrane. These findings were similar to the previous studies that reported fibronectin localization along the electron-dense band of Descemet's membrane and in the stroma of porcine cornea . Further experimental and clinical studies reported fibronectin expression in rabbit cornea and basement membrane of the central cornea of the normal human eyes, respectively [13,14]. The presence of fibronectin in cornea was of clinical importance in promoting corneal healing by providing sites for epithelial cell attachment and migration . It was reported that topical autologous fibronectin healed 16 of the 20 persistent corneal ulcerations resistant to conventional therapy . Parallel to these observations, in-vitro studies using animal models of scrape injured rabbit corneas reported that addition of fibronectin increased migration of epithelial cells. Furthermore, the stimulatory effect of epidermal growth factor on corneal epithelial cell migration was dependent on fibronectin. Other investigators also added that wounding of the cornea induced production of several ECM proteins, such as fibrin and fibronectin .
As demonstrated in this study, fibronectin was expressed around trabecular spaces of Fontana and around the endothelium of Schlemm's canal. This was similar to the previous studies that reported the same results in enucleated human eyes within 12–30 h postmortem . Other investigators also reported that fibronectin is secreted by the trabecular mesh and is considered to be a major ECM component in the trabecular meshwork and Schlemm's canal system . The clinical significance of fibronectin in these areas was attributed to its involvement in trabecular cell–matrix interaction and in intraocular pressure regulation . Abnormal deposition of ECM material, including fibronectin, in the aqueous outflow system was believed to account for the increased aqueous outflow resistance and glaucoma formation [21,22]. Other investigators also added that accumulation of fibronectin was a characteristic feature of aging trabecular cells .
Morphometric analysis revealed maximal fibronectin expression in the sclera. This might be attributed to its protective function and its provision of suitable attachment sites for muscles and resistance to intraocular pressure.
As shown in this study, fibronectin staining was observed in the basement membrane of the bulbar conjunctiva. This was in agreement with other former studies that were carried out on human conjunctiva [24–26]. Clinical studies have observed increased fibronectin deposition in conjunctival thickening of pterygium .
Light microscopic examination of the lens displayed fibronectin staining in its capsule. Fibronectin expression in the lens differs according to animal species. It was evident throughout the adult mouse posterior capsule . Other investigators observed its expression on the outer surfaces of the anterior and equatorial capsules of adult rats  and humans . In contrast, other investigators could not verify fibronectin in purified extracts of calf anterior capsules. This discrepancy might be attributed to quantitative and/or qualitative species differences and to technical differences . The source of fibronectin in the capsule was supposed to be derived from soluble fibronectin of the vitreous and/or of the aqueous humor. Several studies tried to understand the functional role of fibronectin in the eye lens. They reported that its existence was essential as a structural component of basement membranes and as a cell signaling molecule capable of binding both heparin sulfate proteoglycans and collagen IV in the capsule, as well as integrin receptors in the lens cell membranes [31,32]. It has been also proposed that fibronectin might play a role in lens wound healing and posterior capsule opacification . It was reported that fibronectin promoted epithelial cell attachment and migration of lens epithelial cell explants from young embryonic rats , rabbit lenses , and adult human . Clinical studies reported a decrease in fibronectin staining in the human lens during aging and cataractogenesis .
The expression of fibronectin in the choroid at Bruch's membrane and around blood vessels was parallel to several previous studies [38,39]. Data obtained suggested that the presence of normally differentiated retinal pigment epithelium producing fibronectin was necessary for the formation of choriocapillary layer of the choroid in chick embryos , and its existence in this layer was related to chorioretinal wound healing after laser photocoagulation . Other investigators also added that fibronectin anchors the retinal pigment epithelium to the basal lamina through 5β1 integrins .
Retina displayed fibronectin deposition in the outer and inner plexiform layers and prominently in the internal limiting membrane. Coinciding with these results, many investigators observed fibronectin immunostaining in blood vessel walls and in all basement membranes, including the internal limiting membrane of the retina of human and bovine eyes. They also added that fibronectin was arranged in fine fibrillar arrays within the vitreous body [10,29,42]. In contrast, other investigators observed negative fibronectin staining in the inner limiting membrane of the retina in all animals studied . The presence of fibronectin in this membrane, which formed the innermost layer of retina and the outer boundary of vitreous, provided attachment of the vitreous to the internal limiting membrane and of the internal limiting membrane to the Mueller cell processes . Moreover, increased synthesis and deposition of fibronectin by microvascular cells might modify the interactions of cells and matrix with functional consequences relevant to the lesions of retinopathy [41,44].
From this study, it could be concluded that fibronectin displayed wide distribution in the ocular tissues of adult male albino rat. It is abundant in basement membrane, connective tissue stroma, and around blood vessels. It is strongly expressed in the sclera, followed by ciliary body, choroid, iris, cornea, conjunctiva, retina, and finally lens capsule. The clinical relevance of the different distribution of fibronectin may be the subject of further researches.
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