The expressing plasmid vector pRc/CMV, carrying the mouse BDNF cDNA in the
XbaI site, was provided by Yves-Alain Barde (Max-Planck Institute of Neurobiology, Martinsried, Germany).
14 15 The vector was transformed into
Escherichia coli JM109 competent cells (Takara Shuzo, Kyoto, Japan) by heat shock, multiplied by culturing the host
E. coli cells, and isolated with use of a kit (EndoFree Plasmid Maxi Kit; Qiagen, Santa Clarita, CA). The isolated host
E. coli were suspended in 10 mM Tris-HCl buffer (pH 7.4) with 1 mM EDTA (TE buffer; Qiagen) at a concentration of 2.5 μg/μL, as determined by a spectrophotometer (model DU640; Beckman Instruments, Fullerton, CA). For confirmation, the product was digested by
XbaI and electrophoresed on a 0.6% agarose gel before use. The
BDNF gene was approximately 700 to 800 bp, as measured by a 100-bp DNA ladder (data not shown).
In rats under deep anesthesia (intraperitoneal injection of 500 mg urethane, 11 mg ketamine, and 14 mg xylazine per kilogram body weight), the left eye and ON were exposed. The intraocular pressure was first reduced by paracentesis. A small hole was made with a 31-gauge needle 0.5 to 1.0 mm posterior to the limbus, and while the tip of the needle was viewed as it entered the vitreous at a 40° to 50° angle through the dilated pupil, 10 μg plasmid DNA in 4 μL TE buffer was injected into the vitreous.
16 17
The eye was then immediately grasped between forceps-type electrodes, with the cathode attached to the corneal electrode and the anode attached to the scleral electrode, as reported.
13 17 Square-wave pulses of 99-ms duration were applied with five pulses of 12-V/cm field strength, administered twice with a 5-minute interval (Electro Square Porter T820; BTX, San Diego, CA). The rats receiving the
BDNF gene by electroporation are referred to as the BDNF E(+) group. Rats injected with 4 μL phosphate-buffered saline (PBS) or plasmid pRc/CMV with electroporation but containing no
BDNF cDNA are referred to as the PBS group and Plasmid group, respectively, and those receiving 4 μL of
BDNF gene without electroporation are the BDNF E(−) group. Rats that did not receive any intravitreal injection served as the control and are referred to as the NO group.