Infusion syringe pump

Adult cortical injection groups (LV-PGK-α6integrin-eYFP, n=3; LV-PGK-α9integrin-eYFP, n=3; LV-PGK-eGFP, n=5; AAV5-CAG-α9integrin-V5, n=5; AAV-CAG-β1integrin-GFP, n=6) sustained a single injection of 1μl LV or AAV into the left forelimb sensorimotor cortex (SMC) at (AP 1.5 mm, ML 1.5 mm, DV −1.5mm; Krajacic et al., 2009 (link)). Red nucleus injection groups (LV-PGK-α6integrin-eYFP, n=3; LV-PGK-α9integrin-eYFP, n=3; LV-PGK-eGFP, n=3) sustained a single injection of LV into the left red nucleus at (AP −5.9 mm, ML 0.7 mm, DV −7.0 mm). Injections were performed using a custom made 30 gauge stainless steel needle attached to a Hamilton syringe driven by an infusion syringe pump (World Precision Instruments) at 0.2 μl/min. One microliter was injected into the left SMC (LV or AAV) or into the left red nucleus (LV) over a 5 min period, followed by a 3 min period before needle withdrawal.

All animal surgeries were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. In this study, adult two-month-old male Lewis rats were used. DRG virus injection was performed according to the protocol described (Cheah et al., 2017 ). The three viruses used in this study were AAV5-CMV-fGFP, AAV5-CAG-α9-V5, and AVV5-CMV-kindlin1-GFP, and they were obtained from the previous integrin study (Cheah et al., 2016 (link)). Briefly, one microliter of the virus at a working titer of 2 × 10¹² GC/ml was injected into the left C5–C8 DRGs using a custom-made 33-gauge needle syringe (Hamilton) with an infusion syringe pump (World Precision Instruments) at 0.1 μl/min. For the groups of animals with crush injury, the left C5–C8 dorsal roots were crushed using a pair of Bonn forceps (Fine Science Tools) for 3 × 10 s for each root. The animals were kept for eight weeks for recovery, and were culled by exposure to a rising concentration of CO₂. The virus-injected left C5–C8 DRGs were harvested for fluorescence-activated cell sorting (FACS). The spinal cords were fixed with 4% paraformaldehyde (PFA) for post hoc immunohistochemical analysis.

Stereotaxic surgery was performed as described previously^{43 (link)}. Briefly, the mice were anesthetized with 1.5% isoflurane inhalation and stabilized on a stereotaxic instrument (David Kopf Instruments). The location for cerebellum injection was determined according to the distance from bregma: anterior–posterior = −6.3 mm, medial–lateral = ±1.7 mm, dorsal–ventral = −1.5 mm. For striatum: anterior–posterior = 0.55 mm, medial–lateral = ±2 mm, dorsal–ventral = −3.5 mm. For prefrontal cortex: anterior–posterior = 2 mm, medial–lateral = ± 0.25 mm, dorsal–ventral = −1.8 mm. Small holes were drilled in the skull, and a 30-gauge Hamilton microsyringe connected to a syringe infusion pump (World Precision Instruments, USA) was used to deliver the viruses at a speed of 200 nl/min. AAVs were injected unilaterally or bilaterally into the brain regions as indicated. Meloxicam at 5 mg/kg was given before surgery as an analgesic, and the mice were placed on a heated blanket to recover from the anesthetic after surgery. Four to five mice per group were used for stereotaxic injection.