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ejp176-sup-0001-si.pptx497KFigure S1. Measurement of weight bearing (WB) using the Tekscan® system. (A), Architecture of a Tekscan® sensor mat. The sensor mat is made up of two thin flexible polyester sheets with electrically conductive electrode leads placed on top of each other and a semiconductive ink in between creating a grid-like pattern with sensing location (sensel) at each intersection. Pressure applied on top of the sensor mat can be quantified by measuring changes in current flow at each intersection point due to displacement of the semiconductive ink by the pressure. (B), Two Tekscan® sensor mats were joined together in order to accommodate free movement of the rat. (C), Color-coded pressure distribution pattern exerted by different body parts of a cartoon rat touching the Tekscan® sensor mat. Pressure-intensity color coding scale is shown in the lower panel. Tekscan® is a registered trademark of Tekscan Inc, Boston, USA.
ejp176-sup-0002-si.pptx37KFigure S2. Drug administration schedule for each weekly testing in the Tekscan® system. The rats were tested after the first drug administration (acute effects) and following 4 days of twice daily (b.i.d.) drug administration (chronic effects). Similar dosing and testing schedules were followed during week1, week2, week3 and week4 of the MIA model for examining the acute and chronic effects of dexamethasone, celecoxib and duloxetine. Effects of naproxen were examined during week-1 and week-3 after MIA injection using similar dosing and testing schedule. For pregabalin acute effects were examined during week1 and chronic eeffects were examined during week3 using similar dosing schedule. For morphine, drugs were injected once daily and rats were tested for acute and chronic effects during week1 and week3 of MIA model.
ejp176-sup-0003-si.pptx110KFigure S3. Effects of morphine and pregabalin on WB deficits in MIA rats during week3 after MIA injection. (A, B and C), Raw Tekscan® WB trace representing pressure distribution pattern by four limbs and tail of MIA rat treated with vehicle saline, morphine (3 μmol/kg, s.c.) or pregabalin (408 μmol/kg, p.o.). Percent of total body weight borne by each body parts are shown inside the rectangular boxes. Note that rat treated with pregabalin placed an unusually extra weight in the tail/back without any improvement in WB by the MIA-injected left hind paw. (D), Acute effects of morphine and pregabalin on WB distribution pattern on the four limbs and the tail in MIA rats. Note that only morphine (3 μmol/kg, s.c.) was able to reverse WB deficits in the MIA-injected left hind limb (LH), while pregabalin at 408 μmol/kg, p.o., induced an unusual increase in WB in the tail. The rats were tested at 30 min after morphine injection and at 1 h after pregabalin administration. LH, left hind paw; RH, right hind paw; RF, right front paw; LF, left front paw. Please see Table 1 for the % of effects exerted by each drug.
ejp176-sup-0004-si.docx14KAppendix S1. Determination of PGE2 in Synovial fluid Using Liquid Chromatography Tandem Mass-Spectrometry. The samples were thawed on ice and 25 μL of sample was pipetted to a 96 well polypropylene plate (200 μL conical shaped wells). The polypropylene plate was kept on ice. The labelled working solution of deutherium-PGE2 was mixed with ZnSO4 (0.2 M), in the ratio of 3:7. 50 μL of this mixture was added to each well containing the unknown samples. The plate was put on a shaker for 1 min and centrifuged at 2800 g for 30 min. The supernatants (50 μL) were transferred to fresh wells in the same or a new polypropylene 96 well plate. The samples were diluted by 75 μL of 10% acetonitrile in 0.1% formic acid. The plate was put on a shaker for 2 min and then put in the autosampler. Chromatographic measurements were then done using Quantum triple-stage mass spectrometer (ThermoFisher) and Accela ultra high pressure pump (ThermoFisher) and, using a previously constructed standard curve made with 500, 100, 20, 4, 0.8 and 0.16 nM concentrations of PGE2. Both PGE2 and the labelled PGE2 (deutherium-PGE2) were from Cayman Chem. PGE2 stock solution was prepared by weighting 0.261 mg of PGE2 and dissolved in 1 mL of methanol. The deutherium-PGE2 stock solution was prepared by dilution to 1 mL in methanol of 10 μL of 100 ug/200 μL deutherium-PGE2. 20 μL of the stock solution was diluted to 10 mL in methanol to make a labelled working solution of 28 nM deutherium-PGE2.

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