Nanofil microsyringe

Adeno-associated virus (rAAV5EF1a-DIO-hChR2(E123T/T159C)-mCherry) was packaged and purified by the Gene Therapy Center Vector Core at The University of North Carolina at Chapel Hill and estimated by dot blot to be at a concentration of 4X10e12 virus molecules/ml.
Mice were anesthetized by injection (i.p., 10 ml/kg) with a mixture of ketamine (120 mg/kg) and xylazine (16 mg/kg) in saline. Mice were mounted onto a stereotactic frame and small burr holes were made bilaterally (AP = 0.9 mm and ML = −1.9 mm and +1.9 mm, relative to bregma). A NanoFil microsyringe (World Precision Instruments) was lowered to deliver 0.5 µl of virus solution at each of two sites on each side of the brain (DV = 2.0 mm and 2.7 mm), at a rate of 0.1 µl/min. Eight weeks after surgery, mice were transcardially perfused and brain sections were obtained for immunohistological examination as described below.

The procedure for IVT injection has been described previously [29 (link),75 (link),76 (link)]. Briefly, prior to injection animals were anesthetized with 150 mg/kg body weight (BW) ketamine hydrochloride (Parnell Laboratories, New South Wales, Australia) and 10 mg/kg BW xylazine hydrochloride (Troy Laboratories, New South Wales, Australia) and their pupils were dilated with a topical application of 1% tropicamide and 2.5% phenylephrine hydrochloride (Alcon Laboratories Inc., Fort Worth, TX, USA). For the injection, a 1 µL aliquot of solution containing each chemical was injected into the vitreous body of both eyes of each mouse using a Nanofil microsyringe with a 33G needle (World Precision Instruments Inc., Sarasota, FL, USA), under an ocular surgery microscope (Leica Microsystems GmbH, Wetzlar, Germany). A bilateral approach was taken to minimize the number of animals used in this study. All procedures were performed carefully to avoid injuring the lens or retina. Four or seven days following injections, the eyes were collected and subjected to further analysis as described below. Eyes with bleeding or inflammation caused by technical errors were excluded from analysis.

We utilized the AAV Helper-Free System (AAV Helper-Free System, Stratagene) for viral production using a triple-transfection, helper-free method and purified it as described in a previous study. The interference sequences were as follows: Smad7 shRNA: 5ʹ-CACCGCTTTCAGATTCCCAACTTCTCGAAAGAAGTTGGGAATCTGAAAGC-3ʹ and control shRNA, 5′-TTCTCCGAACGTGTCACGT-3′. Mice were anaesthetized with an isoflurane/air mix (3% for initial induction and 1.5-2% for maintenance). Three hundred nanoliters of either AAV5-shSmad7 or AAV5-shCtrl was injected into the subcutaneous of mice dorsal skin 5 days before mechanical loading and continuously injected during the loading period according to different groups. The injections were performed using a 34-gauge needle (World Precision Instruments) attached to a 10-μL NanoFil microsyringe (Nanofil, World Precision Instruments).