To further investigate the pathologic roles of hemopexin in VKC, an allergic conjunctivitis mouse model, according to the protocol of Nakamura et al, was used to study the anatomic and cellular responses on the ocular surface.12 Animal ethics approval was obtained from The Chinese University of Hong Kong, and all animal care was carried out according to the institutional guidelines. Briefly, 8-week-old Institute for Cancer Research mice were randomized and systemically sensitized by an intraperitoneal injection of 0.1 mL of 10-μg/mL recombinant hemopexin protein (R&D Systems, Minneapolis, Minnesota) or vehicle (sterile phosphate-buffered saline solution) on alternate days for 2 weeks (day 0 to day 14). Short ragweed (SRW) pollen extracts (Greer Laboratories, Inc, Lenoir, North Carolina), at 100 μg/mL in 0.1 mL, were used for comparison because SRW has been used for genetic and immunologic studies in an allergic conjunctivitis model.11 The sensitization period was followed by administration of high-titer topical eyedrops of recombinant hemopexin protein (100 μg/mL in 0.02 mL) or SRW (400 μg/mL in 0.02 mL) to the left eye once daily for a week (day 13 to day 21) until the appearance of mild or moderate clinical symptoms. The right eye was administered normal saline as a control in the same animal. To avoid crossover, the mice were maintained in a lateral position during topical administration, and the volume was controlled to 5 μL in each drop 4 times during 5 minutes. Drop administration was completed in one eye and then in the other eye 30 minutes later. The experimental challenge of higher titers of hemopexin or SRW was performed on day 24. All animals were assessed by masked observers. Both the behavior of mice in the cage and specific clinical symptoms in each eye, such as tearing and discharge, conjunctival edema and redness, and lid edema and redness, were assessed. To evaluate the inflammatory cell infiltration in the conjunctiva during the late-phase reaction, whole eye tissues were excised 24 hours after the final challenge (day 25), fixed in 4% paraformaldehyde and embedded in paraffin wax for sagittal sectioning, standard histologic staining, and immunohistochemical analysis, as described in the “Impression Cytology and Immunocytochemistry” subsection. Particular attention was paid to anatomic changes in the cornea, conjunctival complications, and eyelid inflammation.