الفهرس 
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Abstract
Chemical injury of the eye is a true ocular emergency that requires immediate intervention. Alkaline injuries are more severe than acid injuries. Alkaline injuries to the eye can produce extensive damage to the cornea leading to scarring with affection of visual acuity. Exosomes are effective in treating various diseases and do not have the drawbacks of mesenchymal stem cells (MSCs). Therefore, the attention of researchers was directed towards using exosomes as cell free therapy in treating various diseases.
This study was carried out to study the possible effect of MSCs-derived exosomes on healing of experimentally induced corneal alkali burn in adult male New Zealand white rabbits by histological and immuno-histochemical techniques.
Forty-five adult male New Zealand white rabbits -each weighing between 2-2.5 kg- were included in this study. After acclimatization period, animals were randomly divided into three groups: group I served as a control group, and it was further subdivided into two equal subgroups: Subgroup Ia (negative control) and subgroup Ib (positive control). In both subgroups, half rabbits were sacrificed after seven days, and the other half were sacrificed after 14 days. group II served as corneal alkali burn group in which right corneal alkali burn was induced using topical sodium hydroxide. For examination of early corneal alkali burn, five rabbits were sacrificed after one hour from induction of alkali burn. The remaining rabbits were left for spontaneous corneal healing, then they were further subdivided into two subgroups: Subgroup IIa and subgroup IIb, in which rabbits were sacrificed after seven days and 14 days (respectively) from the beginning of the experiment. group III served as exosomes treated group, in which corneal alkali burn was induced using topical sodium hydroxide. After one hour of corneal alkali burn induction, rabbits were given a sub-conjunctival injection of phosphate buffer saline containing purified MSCs- exosomes. Subconjunctival injection of exosomes was repeated every other day till the end of the experiment. This group was further subdivided into two subgroups: Subgroup IIIa and subgroup IIIb, in which rabbits were sacrificed after seven days and 14 days (respectively) from the beginning of the experiment.
characterization of exosomes was done using transmission electron microscope. Gross examination of the right corneal alkali burn was done to all rabbits using fluorescein dye strips (on days 1, 7 and 14) and hand-held light (on days 7 and 14). At the end of the experiment, right corneal specimens were collected and subjected to Hematoxylin and eosin (H&E), periodic acid Schiff (PAS), and Masson`s trichrome. Immunohistochemical technique was also done using anti-rabbit Transforming growth factor-beta (TGF-β), and anti-rabbit vascular endothelial growth factor (VEGF). Histomorphometric study and statistical analysis were also performed.
In the current study, gross examination of the right cornea -using fluorescein dye strips- showed faster healing in group III on days 1, 7, and 14 compared to corresponding days in group II. Moreover, the pupil was visualized in group III at day seven, while it could not be detected in all examined rabbits of group II at all time intervals.
Regarding Gross examination of the right eye in subgroup IIa using hand-held light at day seven, complete corneal opacity, as well as redness of the conjunctiva were seen. While in subgroup IIIa, transparent cornea was noticed in most rabbits. Minimal redness of the conjunctiva was still observed. On the other hand, Gross examination of the right eye in subgroup IIb using hand-held light at day 14 showed minimal regression of corneal opacity. Moreover, redness of the conjunctiva as well as increased vasculature around the whole perimeter of the limbus were also seen. While in subgroup IIIb, marked improvement was noticed with the appearance of transparent cornea in all examined rabbits. Absence of conjunctival redness and blood vessels was also noticed.
Examination of H&E-stained sections of early alkali burn showed desquamation of corneal epithelium with loss of demarcation between the epithelium and stroma. Corneal stroma appeared with widely separated collagen fibers. PAS-stained sections showed no abnormalities, while Masson’s trichrome stained sections showed irregularly arranged widely separated collagen fibers in the stroma.
On day seven, H&E-stained sections of subgroup IIa showed denuded epithelial surface, superficial keratinization, appearance of pyknotic and karyolitic nuclei. The stroma was seen with widely separated loosely packed sometimes disrupted collagen fibers. Mononuclear infiltration, neovascularization and extravasation of red blood corpuscles were frequently seen. Descemet`s membrane was seen separated from the stroma and corneal endothelium was seen vacuolated. While in subgroup IIIa, improvements of most histological changes were observed. Areas of widely separated collagen fibers, neovascularization and extravasation of red blood corpuscles were occasionally seen in the stroma.
Examination of PAS-stained sections of subgroup IIa showed significant increase in thickness of Descemet’s membrane with areas of focal disruption, as well as increased PAS reaction in some stromal fibers. In subgroup IIIa, although Descemet`s membrane still appeared thick, focal disruption and separation between the membrane and the stroma were not seen. In addition, the stroma appeared with homogenous weak PAS reaction.
Masson’s trichrome stained sections of subgroup IIa showed irregularly arranged widely separated collagen fibers in the stroma with areas of segmental red staining of collagen fibers. In contrast, subgroup IIIa, showed regular almost closely packed collagen fibers.
Immuno-stained sections for TGF- β, in subgroup IIa showed moderate reaction in some cells of corneal stroma, while in subgroup IIIa, mild positive reaction was occasionally noticed in few cells of corneal stroma. Immuno-stained sections for VEGF, in subgroup IIa showed strong positive immune reaction in the endothelium of stromal neovascularization. While moderate reaction was noticed in some stromal cells near the newly formed vasculature. On the other hand, in subgroup IIIa, weak reaction was seen in the endothelium of neovascularization that was occasionally seen in the corneal stroma.
On day 14, H&E-stained sections of subgroup IIb showed disturbed arrangement of corneal epithelium with areas of disruption of epithelial basement membrane and separation of the epithelium from the underlying stroma. Fibrous scars were frequently noticed in the superficial part of the stroma. Neovascularization and extravasation of red blood corpuscles were still observed in the stroma. Appearance of collagen fibers behind the Descemet’s membrane and thickening of endothelial cells were also noticed. In contrast, subgroup IIIb showed improvement of most histological changes but slight irregularity of basement membrane of corneal epithelium was noticed.
With examination of PAS-stained sections of subgroup IIb, Descemet`s membrane appeared with areas of focal disruption. Strong PAS reaction was also noticed in some stromal fibers. While in subgroup IIIb, absence of focal disruption of Descemet’s membrane as well as the weak PAS reaction in stromal fibers were noticed.
Masson’s trichrome stained sections of subgroup IIb showed widely separated collagen fibers with loss of their normal packed appearance. Moreover, collagen fibers that appeared behind the Descemet’s membrane were stained red. In contrast, subgroup IIIb showed regular closely packed collagen fibers.
Immuno-stained sections for TGF- β, in subgroup IIb showed strong reaction in many cells of corneal stroma, while in subgroup IIIb, negative immune reaction was seen in the stromal cells. Immuno-stained sections for VEGF, in subgroup IIb showed strong reaction in the endothelium of stromal neovascularization, as well as in stromal cells. In contrast, subgroup IIIb showed mild immune reaction in the endothelium of few stromal neovascularization.
Such findings revealed that -compared to group II- group III showed faster re-epithelialization of the cornea, enhanced formation of well-defined basement membrane without being separated from the epithelium or the stroma, better organization of collagen fibers in corneal stroma, prevention of inflammation and neovascularization, and proper healing of Descemet’s membrane, as well as preservation of corneal endothelial structure. Hence, we concluded that early and repeated sub-conjunctival injection of MSCs-derived exosomes could be effective in the healing of corneal alkali burn in adult rabbits.