Two groups of rabbits received injections of 0.05 mL of V-MPL (1 mg/mL) suspension, or 0.05 mL of MPL (1 mg/mL) suspension into their right eyes. Subsequently, at 0.5, 1, 3, 6, 12, 24, 48, 72, and 168 hours, injected eyes (six rabbits/interval/group) were clinically graded by a board-certified ophthalmologist (blinded to treatments) using a slit lamp and indirect ophthalmoscopy with pupil dilation (2.5% phenylephrine and 0.5% tropicamide). Slit lamp examination was used to rate the following: (1) blepharitis (i.e., eyelid margins with injection, suppuration, and hair loss); (2) conjunctivitis/scleral injection (i.e., conjunctiva and sclera with injection, hyperemia, and chemosis); (3) limbal injection (i.e., the degree of peripheral redness near the cornea); (4) iritis (i.e., color changes, change ratios, and light reactivity of the iris); (5) corneal clarity (i.e., the opaqueness resulting from infection); (6) hypopyon (i.e., the degree of inflammatory cells in the anterior chamber); (7) fibrin production (i.e., the amount of fibrin in the anterior chamber); and (8) anterior chamber cells (i.e., the number of cells and flare seen with the slit lamp light beam at 2 mm). Indirect ophthalmoscopy with pupil dilation was used to rate inflammation of the vitreous and any retinal changes, as follows: (9) vitreous grade (i.e., ability to visualize the retina clearly); and (10) the retina with clear vitreous (i.e., the presence of abnormalities within the retina itself). All clinical manifestations were rated on a scale of increasing severity: 0, 0.25, 0.5, 1, 2, and 3, with a maximum score of 30.
After clinical ratings were completed, aqueous humor was extracted, vancomycin concentrations were determined by the same HPLC dosage procedure as in experiment 2, and intraocular vancomycin concentrations were calculated as changes over time.
The eyes were then carefully dissected from their orbits, and the corneas were cut under a laminated flux hood and placed in cornea prep medium (Eurobio, Les Ulis, France). Residual eye tissue was immersed in formaldehyde for histology of primarily the iris, ciliary body, and retina.
The determination of the density and viability of the corneal endothelial cells was performed at an eye bank, with the same procedure as that used for human corneas and in accordance with the recommendations of the European Eye Bank Association (EEBA, 2006). The eye bank was not aware of the time after injection, the age of the rabbits, or any other experimental data. Corneal endothelial cell density was determined by optical microscopy using a 1-mm2, 10 × 10-square graticule for direct counting (Leica Biomed microscope; Reichert, Vienna Austria, with CCD camera; Sony, Tokyo, Japan) after staining with trypan blue (0.4% sterile trypan blue; Edouard Herriot Hospital, Lyon, France) for 1 minute, and intercellular space dilation for 4 minutes with 0.9% wt/vol apyrogenic sterile NaCl (Aguettant, Lyon, France). The count was performed on two 1-mm2 zones: one located in the central cornea and the other 2 mm away from the central point (within an 8-mm diameter). Results are expressed as the average cell count in the two zones (cells/mm2). Apoptotic or dead cells retained trypan blue staining in their nuclei, whereas live cells eliminated the stain by active transport systems. Dead cells were counted in each of the 1-mm2 areas.
Toxicity in the remaining eye tissues (excluding the cornea) was established by histology, conducted according to the following procedures. Each globe was formalin fixed, paraffin embedded, and sectioned horizontally through the optic nerve. A representative HES (hematoxylin, eosin, saffron) 3-μm section was then analyzed (treatments blinded) by light microscopy. Inflammation intensity was scored as: − (none), + (slight), ++ (mild), and +++ (severe).