The healing response in corneas after RGX is very similar to that observed after DE alone, suggesting that, along with its short treatment time and limited effect on keratocytes, RGX displays good potential for clinical cornea stiffening.
These results indicate that corneal stiffness increases after CXL, and further increases as a function of time after both RGX and UVX. Also, while biomechanical properties determined after ex vivo CXL are indicative of corneal stiffening, they may not provide entirely accurate information about the responses to CXL in vivo.
Pollen is the most common aeroallergen to cause seasonal conjunctivitis. The result of allergen exposure is a strong Th2-mediated response along with conjunctival mast cell degranulation and eosinophilic infiltration. Oleanolic acid (OA) is natural a triterpene that displays strong anti-inflammatory and immunomodulatory properties being an active anti-allergic molecule on hypersensitivity reaction models. However, its effect on inflammatory ocular disorders including conjunctivits, has not yet been addressed. Hence, using a Ragweed pollen (RWP)-specific allergic conjunctivitis (EAC) mouse model we study here whether OA could modify responses associated to allergic processes. We found that OA treatment restricted mast cell degranulation and infiltration of eosinophils in conjunctival tissue and decreased allergen-specific Igs levels in EAC mice. Th2-type cytokines, secreted phospholipase A2 type-IIA (sPLA2-IIA), and chemokines levels were also significantly diminished in the conjunctiva and serum of OA-treated EAC mice. Moreover, OA treatment also suppressed RWP-specific T-cell proliferation. In vitro studies, on relevant cells of the allergic process, revealed that OA reduced the proliferative and migratory response, as well as the synthesis of proinflammatory mediators on EoL-1 eosinophils and RBL-2H3 mast cells exposed to allergic and/or crucial inflammatory stimuli such as RWP, sPLA2-IIA or eotaxin. Taken together, these findings demonstrate the beneficial activity of OA in ocular allergic processes and may provide a new intervention strategy and potential therapy for allergic diseases.
PURPOSE. To study corneal wound healing after two cross-linking techniques using either rose bengal and green light (RGX) or the conventional treatment using riboflavin and UVA radiation (UVX). METHODS. Corneas of New Zealand rabbits were monolaterally treated with UVX (21 eyes) or RGX (25 eyes). Treatments involved corneal de-epithelialization (8-mm diameter), soaking with photosensitizer (0.1% riboflavin in 20% dextran for 30 minutes for UVX; 0.1% rose bengal for 2 minutes for RGX), and light irradiation (370 nm, 3 mW/cm 2 , 30 minutes for UVX; 532 nm, 0.25 W/cm 2 , 7 minutes for RGX). Contralateral eyes were used as controls. Clinical follow-up included fluorescein staining, haze measurement, and pachymetry. Healing events analyzed after euthanasia at 2, 30, and 60 days included cell death (TUNEL assay), cell proliferation (BrdU [bromodeoxyuridine] immunofluorescence), and differentiation to myofibroblasts (a-SMA [alpha smooth muscle actin] immunohistochemistry). RESULTS. Re-epithelialization and pachymetries were similar after RGX and UVX. The haze from day 1 to 15 was greater after UVX. Cell death was deeper after UVX, being localized in the anterior and middle stroma, and was superficial (anterior third) after RGX. Cell proliferation appeared after 2 days and was localized in the middle and posterior stroma in the UVX group but was superficial in the RGX group. After 60 days the number of stromal cells had not returned to the control number in either group. CONCLUSIONS. The deeper and longer-lasting cell damage caused by UVX compared to RGX may underlie the slower cell repopulation after UVX and other differences in healing. Shallower damage and a shorter treatment time suggest that RGX may be appropriate for stiffening thin corneas.
The extracellular matrix (ECM) confers transparency to the cornea because of the precise organization of collagen fibrils and a wide variety of proteoglycans. We monitored the corneal wound healing process after alkali burns in rabbits. We analyzed the location and expression of collagens and proteoglycans, the clinical impact, and the recovery of optical transparency. After the animals received both general and ocular topical anesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. The eyes were evaluated under a surgical microscope at 1, 3, and 6 months after burning. At each time point, the clinical conditions of the burned and control corneas were observed. The arrangement of collagen fibers in the corneal stroma was visualized by Picrosirius-red staining, Gomori's silver impregnation and transmission electronic microscopy. Corneal light transmittance was also measured. Myofibroblasts presence was analyzed by immunohistochemistry. mRNA expression levels of collagen types I and III, lumican, decorin, keratocan and alpha-smooth muscle actin were determined by quantitative real-time polymerase chain reaction. One month after alkali burn, the ECM was disorganized and filled with lacunae containing different types of cells and collagen type III fibers in the wound area. Corneal opacities were present with attendant loss of light transmittance. Collagen and proteoglycan mRNA expression levels were up-regulated. After three months, wound healing progress was indicated by reduced corneal opacity, increased light transmittance, reorganization of collagen fibers and only collagen type I expression levels were at control levels. After six months, the wound area ECM morphology was similar to controls, but transmittance values remained low, denoting incomplete restoration of the stromal architecture. This multidisciplinary study of the stromal wound healing process revealed changes in corneal transmittance, collagen organization, myofibroblasts presence and ECM composition at 1, 3, and 6 months after alkali burning. Documenting wound resolution during the six-month period provided reliable information that can be used to test new therapies.
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