All mice were purchased from Jackson Laboratory (The Jackson Laboratory; Bar Harbor, ME), and research protocols conformed to the standards of the Association for Research in Vision and Ophthalmology for the Use of Animals in Ophthalmic Care and Vision Research, as well as the Boston University IACUC. The C57BL/6J mice were fed either a control diet (B6 control) or a high fat diet to induce obesity (DiO) (Jackson Laboratory): Control Diet D12450B (10 kcal% fat, 3.8 kcal/g) and High Fat Diet D12492 (60 kcal% fat, 5.2 kcal/g) [6 (link)].
To examine localization of P2X7 and pannexin1 in response to injury, 1.5 mm debridement wounds were performed on both B6 control and DiO mice, as previously described [6 (link), 17 (link), 18 (link)]. After debridement, the mice were euthanized, the eyes enucleated, and placed in organ culture [12 (link), 17 (link)], and incubated in serum-free Keratinocyte medium (Invitrogen Carlsbad, CA) containing 100 μg/mL penicillin and 100 μg/mL streptomycin for 2, 8, or 20 hours at 37°C and 5% CO2. A minimum of 3 eyes per time point per condition was analyzed.
To examine localization of P2X7 and pannexin1 in response to injury, 1.5 mm debridement wounds were performed on both B6 control and DiO mice, as previously described [6 (link), 17 (link), 18 (link)]. After debridement, the mice were euthanized, the eyes enucleated, and placed in organ culture [12 (link), 17 (link)], and incubated in serum-free Keratinocyte medium (Invitrogen Carlsbad, CA) containing 100 μg/mL penicillin and 100 μg/mL streptomycin for 2, 8, or 20 hours at 37°C and 5% CO2. A minimum of 3 eyes per time point per condition was analyzed.
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