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Technical details of IUE have been described previously⁸⁸ . Here, we summarize key details for IUE of purified plasmid DNA. Electroporation was performed using a square wave electroporator (CUY21EDIT, Nepagene) at E13.5 (labeled as E13 in figures) to target cortical L4 neurons in primary somatosensory cortex. Five square wave pulses (50 ms duration, period 1 s) at 30 V were applied to embryos using round (3 mm) platinum plate tweezer electrodes (Nepagene). After electroporation, the abdominal cavity was sutured, and skin closed with wound clips (AutoClip, Fine Science Tools). All plasmids were used at 1 µg/µL, unless indicated otherwise. Sparse-labeling experiments were performed using either pCAG-LoxP-Neomycin-(STOP)-LoxP (LSL)-EYFP or pCAG-LSL-DsRed2, along with a titrated amount of pCAG-Cre (0.2–1 ng/µL) to allow visualization of distinct neuron morphologies. Sparse BFP-[Histone- GFP]-TVA66T-[N2c Glycoprotein] (BHTG) rabies construct labeling was also achieved with a titrated amount of pCAG-Cre (1–20 ng/µL).

HF was induced by continuous infusion of isoproterenol with an osmotic pump, following the conventional procedure in this model [9 (link)]. Briefly, mice were anesthetized with a mixture of inhaled isoflurane and oxygen-enriched air (1.25% during induction and 1% during maintenance) and a small incision was made on the back of each animal between the shoulder blades after removing the hair from the area using depilatory cream. The skin was carefully separated from underlying connective tissues using blunt-ended scissors and an osmotic mini-pump (Alzet, model 1004) containing isoproterenol (Sigma Aldrich) at 30 mg/kg per day dissolved in sterile 0.9% NaCl solution or only 0.9% NaCl solution (for healthy controls) was implanted subcutaneously for delivery pharmacological agent or placebo for 30 days and the incision was sutured with surgical staples (Autoclip, Fine Science Tools). The procedure was performed under aseptic conditions. The surgery platform was continuously warmed to maintain body temperature until the end of anesthesia. Buprenorphine (0.1 mg/kg) was subcutaneously administered 10 minutes before surgery and after 24 hours. Suture staples were removed 7 days after surgery.

The spared nerve injury (SNI) model of neuropathic pain was used. Baseline values for behavioral experiments were established 24-h prior to surgery. Mice were anesthetized under isoflurane anesthesia (1.0–2.5%). The ipsilateral thigh was shaved and cleaned with betadine (Dynarex, NY, USA; cat no. 1425) and 70% ethanol (Decon Labs, PA, USA; cat no. 2701). The skin and muscle of the ipsilateral thigh were incised with a #11 scalpel (Thermo Fisher, MA, USA; cat no. 22-079-691) and the sciatic nerve along its three branches (common peroneal, tibial, and sural) were exposed. A tight ligature using a 5-0 silk suture (VWR, PA, USA; cat no. MV-682) was placed around the proximal tibial and common peroneal branches, after which the nerves distal to the ligature were transected, taking care to not stretch or damage the sural nerve. The skin was closed using an auto clip (Fine Science Tools, CA, USA; cat no. 12022-09) and mice were returned to their home cages to recover (Decosterd and Woolf, 2000 (link)). Sham surgeries were done identically to the SNI surgery; however, no portion of the sciatic nerve was ligated or transected. Following surgery, mice are subcutaneously administered a single dose of Gentamicin (5 mg/mL) (Sigma-Aldrich, CA, USA; cat no. G1272) as a prophylactic antibiotic. All mice were then returned to their home cages for recovery and monitored daily.