with a final step at 72uC for 7 min. The resulting products were separated by 1% agarose gel electrophoresis and visualized by ethidium bromide staining and using a Fotodyne Trans-illumina- tor (Fotodyne Inc., Wisconsin). Control reactions containing no reverse transcriptase were run simultaneously. Cyclic Mechanical Stretch Porcine TM cells between passages 4 and 5 were plated onto BioFlex six-well plates pre-coated with collagen (Flexcell Interna- tional, Hillsborough, NC). Once they reached confluence, cells were serum starved for 24 hrs and then stretched using the FlexCell FX-4000 system (Flexcell International, Hillsborough, NC). A cyclic stretch of 1 cycle/second was applied to the experimental plates for 17 hrs at 37uC as we described earlier [47]. Control plates were incorporated into the same system, but did not undergo stretch. Experimental plates were exposed to a total stretch of 10% as compared to control plates. After stretch treatment, media were collected, centrifuged (3000 rpm, 4uC for 10 minutes) and supernatants were used for detection of ATX by immunoblotting. The cells were scraped and lysates were prepared for Western blotting analysis as previously described. siRNA Treatment HTM cells (passaged between 3 to 5 cycles; derived from donors aged 16, 18 and 74 years) were cultured in 100 cm plates in DMEM with 10% FBS, until they reached about 80% confluency. Medium (containing 10% FBS) was changed a day before and on the day of transfection. Cells were trypsinized and counted prior to transfection, and 56105 cells were used for each nucleofector transfection. Two different controls: untransfected control, and FITC-conjugated scrambled siRNA control were set up for these experiments. The Amaxa Nucleofector (program T023) and Basic Endothelial Cell Nucleofector Kit (C. No. VP1-1001) were used and the kit specifications were strictly adhered to. Post transfec- tion, cells were plated in 6-well plates and coverslips coated with 2% gelatin for immunostaining. Cells were changed into fresh medium on the day following transfection. FITC fluorescence was detected after 24–36 hours of transfection. Prior to cell lysate preparation for Western blot analysis, cells were changed into serum-free media overnight and the standard procedure was followed for cell lysate preparation. Coverslips were fixed for actin filament staining using TRITC-phalloidin as described earlier. Animal experimentation Topical drug effects. In the first set of experiments, we used four Dutch-Belted adult male rabbits. One eye was treated with drug while the other (contralateral) eye was used as control (sham treatment). Basal IOPs were measured using a pneumotonometer (Model 30, Medtronic) for a week before beginning the experiments to monitor for any irregularities in IOP. The ATX inhibitor (S32826; Sigma) was dissolved in sterile autoclave distilled water (resulting in an alkaline pH,8) to obtain a desired concentration (2–10 mM) for the topical dosing. pH adjusted (pH,8) distilled water served as a control for the contralateral eye. Before administering drug or sham control vehicle, the eyes were treated with a drop of topical anesthetic (0.5% proparacaine HCl). The animals were awake during the IOP measurements. Basal IOPs were measured prior to drug treatments. 35 ml of the drug or sham control vehicle were applied to the respective eyes. This volume was found to be appropriate, covered the entire cornea and did not spill over from the eye. During drug application, the rabbit eyes were kept open physically to prevent blinking for a minute before applying a second aliquot of 35 ml drug solution. In the first set of rabbits, 3 different concentrations of the drug (10, 2 Autotaxin and IOP and 5 mM) were tested in separate experiments (sequentially). Only for the 10 mM concentration, a second dose of drug was administered 5–6 hrs (typically at ,4 PM) after the first dose (typically at ,10 AM). The effects of 10 mM drug were repeated in a second set of rabbits under identical conditions as the first experiment, using 5 additional rabbits. IOPs were measured at 2, 5, 7, 24, 32 and 50 hrs after the first dose of drug application. An average of four readings was used to plot the IOP data for each time point. For the lower concentrations of drug (5 mM and 2 mM), only one morning dose was applied every day (,10/11 AM) for 5 days, and IOPs were monitored twice a day. For the concentration response experiments, animals received sufficient recovery time after each concentration experiment was completed, and the next concentration experiment was tested only after IOP returned to the basal or normal level. The rabbits from the first set of experiments were sacrificed after they received the 5 mM drug for 5 days, after which AH was collected and enucleated eyes fixed for histological analysis. Intracameral drug effects. Due to drug precipitation on the corneal surface and conjunctival hyperaemia associated with topical drug delivery, an intracameral injection experiment was designed using a new batch of four rabbits (same strain as the previous experiments). The volume of AH in adult rabbits was estimated to be ,200–250 ml based on our previous observations. For this study, the drug stock was prepared initially in DMSO and subsequently diluted in PBS, such that the final DMSO concentration in the aqueous humour would be ,0.05%. Prior to intracameral injections, basal IOPs were measured in awake rabbits as described above. The rabbits were anesthetized using intramuscular injections of ketamine and xylazine cocktail, followed by a topical drop of 0.5% proparacaine HCl. An operating microscope was positioned over the eye undergoing surgery and the anterior chamber was entered in the super- otemporal quadrant using a 0.3 cc insulin syringe positioned at an angle and 5 ml of drug (100 mM stock in PBS with 2% DMSO) was injected into the eye anterior chamber. The injections and follow- up ocular changes were performed by a board certified glaucoma specialist. The contralateral eye served as the non-injected control. IOP values in both eyes were measured at 24, 48, 72, 96, and 120 hrs post drug injections, until the pressures in the injected eyes returned to normal. During this time, the eyes were monitored for ocular changes using a slit lamp. Once these rabbits recovered from drug injections after 2 weeks, the contralateral eye was injected with 5 ml of PBS containing 2% DMSO and served as the DMSO-injected sham control. IOPs were measured in both eyes at 24, 48, 72, 96, and 120 hrs post injection. Histology Rabbit eyes from the topical drug experiment (5 mM drug) were enucleated, rinsed in 16 PBS and fixed in 4% paraformal- dehyde (PFA) for 1 hr at room temperature. After this, the lens was carefully removed and the anterior segment was cut into two halves, one half being processed for assessment of corneal toxicity. Briefly, tissue was immersed in 10% buffered formalin at room temperature and sent for pathological analysis, where it was processed and stained with haematoxylin and eosin (H&E). The other half was processed for histological changes at the AH outflow angle. The tissue was immersed in 2.5% glutaraldehyde, 50 mM cacodylate buffer (pH 7.2) containing 4% sucrose and 2 mM CaCl2 for 2 hrs, then transferred to 10% buffered formalin for 48 hrs. The specimens were then embedded in methyl methac- rylate, and 2 mm sections were cut and stained with hematoxylin and eosin stain using conventional methods. Micrographs were captured using a Zeiss light microscope. PLOS ONE | www.plosone.org 12 August 2012 | Volume 7 | Issue 8 | e42627