Diclofenac

Lmnb1 silencing in vivo was carried out by in utero electroporation using Lmnb1 shRNA (Sh-Lmnb1) or scramble-shRNA (Sh-Scr) plasmids as described^{35 (link)}. The pregnant female C57BL6/J mouse (E14) was anesthetized using isofluorane. After injection of DNA (3 µg) containing 1% Fast-Green dye (Sigma) through the uterine wall into the lumen of the telencephalic vesicles, each embryo was subjected to electroporation by five square electric pulses (30 mV for 50 ms, 1 s intervals) delivered through platinum electrodes (Sonidel) using a BTX-ECM 830 electroporator (Harvard Apparatus). After electroporation, the abdominal cavity was sutured and the pregnant mouse was administered diclofenac (0.5 mg/kg) (Voltaren, Novartis) by intraperitoneal injection and kept warm until awake. For Lmnb1 knockdown studies, electroporated pups were born and immediately transferred to a foster CD-1 mother. The pups were sacrificed at postnatal day 7 (P7) and the brains collected for analysis.

The ear edema test model induced by 12-O-tetradeca-noylphorbol-13-acetate (TPA) (Sigma-Aldrich, Saint Louis, MO, USA) was used [48 ]. TPA was dissolved in ethanol (2.5 µg/20 mL), and the solvent did not interfere with the assay. The experimental animals were randomly assigned into three groups: TPA (only positive control), TPA plus diclofenac (Novartis, Mexico City) (nonsteroidal anti-inflammatory drug, negative control) and TPA plus ChEEP. The left ears of the mice were not treated and were the control in each organism. The animals were fasted for 4 h prior to the experiment. Subsequently, they were anesthetized with 250 µL of isoflurane (PISA, Mexico City, Mexico) (inhalation anesthetic), and 10 µL of TPA was administered topically to the inner and outer surface of the right ear of each group. Thirty minutes after the application of TPA, animals received 10 µL of diclofenac or 10 mg of ChEEP on both sides of the right ear. Mice were sacrificed by cervical dislocation after 4 h, and a circular punch biopsy (5 mm) of the right and left ears was taken and fixed in formalin. The histological evaluation included H&E staining and ear thickness was measured with the ImageJ program. Inflammation was assessed by the increase in thickness of the right ear compared to that of the left ear of each of the experimental organisms.