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In!the!present!work,!the!use!of!siRNA,!designed!to!silence!ß2Yadrenoceptors!

has! been! used! in! order! to! reduce! IOP! and! therefore! consider! it! as! a! possible!
strategy!for!the!treatment!of!ocular!hypertension!and!glaucoma.!

2.(MATERIAL(AND(METHODS(
2.1.$Animals$

Male! New! Zealand! rabbits! were! 6Y8! weeks! of! age! at! the! time! of! the!
experiment.! The! siRNAs! were! topically! applied! in! one! eye,! the! contralateral!
receiving! the! same! volume! of! saline.! Each! of! the! treated! eyes! received! an! amount!
of! 250! µg! of! the! siRNA! dissolved! in! 40! µL! of! 0.9! %! NaCl.! The! instillation! was!
performed!in!a!blind!fashion!therefore!no!indication!of!the!saline.!The!siRNA!were!
applied! in! a! blind! fashion! Rabbit! eyes! were! treated! with! 1,1! mg! HPLCYpurified!
siRNAs,! applied.! The! siRNAs! were! applied! in! one! single! eye! in! 265! µg! 0.9%! NaCl!
drops!(40!µL!volume)!on!four!consecutive!days;!the!contralateral!eye!was!instilled!
with!saline!solution!(0.9%!NaCl).!Experiments!were!performed!using!a!single!blind!
design:! no! visible! indication! was! given! to! the! experimenter! as! to! the! applied!
solution! (agent! or! vehicle).! IOP! was! measured! on! ten! days! and,! afterwards,!
animals!were!killed!and!both!eyes!were!collected.!

2.2.$Intraocular$pressure$(IOP)$measurements$in$rabbits$

IOP! was! measured! by! means! of! a! TonoYPen! XL! contact! tonometer! (Mentor!
Massachusetts!Inc.,!Norwell,!MA).!Topical!anesthesia!(Colircusi,!Laboratorios!Cusi,!
Spain:!0.1!mg/ml!tetracaine!plus!0.4!mg/ml!oxybuprocaine!in!0.9%!saline,!diluted!
1:3!in!0.9%!saline)!was!applied!(10!µL)!to!the!cornea!before!each!measurement!to!
avoid!animal!discomfort.!

All!the!siRNA!experiments!were!performed!and!measured!at!the!same!time.!
In!each!series!of!experiments,!IOP!was!measured,!from!the!first!day,!three!times!a!
day!every!four!hours!and,!once!the!IOP!started!to!change!the!measurement!pattern!
moved! once! every! two! hours.! The! siRNA! assays! carried! out! were! purchased! from!
Santa!Cruz!(USA)!(n=8),!and!siRNA!scramble!was!also!from!Santa!Cruz!(USA)(n=4).!

In!the!experiments!performed!with!commercially!available!drugs!(40!µL!of!
commercial!Xalatan,!Trusopt!and!Timoftol),!IOP!levels!were!measured!every!hour!
for!a!total!of!8!hours,!following!the!same!protocol!as!described!before.!!

2.3.$Cell$treatments$and$tissue$sections$and$histology$

Ciliary! body! nonYpigmented! epithelial! cells! were! used! in! order! to! see! the!
silencing! of! the! beta2! adrenergic! receptors.! These! cells! were! kindly! provided! by!
Dr.! CocaYPrados! (University! of! Yale),! and! were! treated! with! an! antibody! against!
the!beta2!adrenergic!receptor!(Ab40834!from!abcam).!!

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