Hemostat

Mice were anesthetized in an induction chamber with 5% (v/v) isoflurane (Zoetis) supplied with oxygen at a flow rate of 0.6 L/min. After lack of a foot pinch reflex, mice were maintained at 2.5% (v/v) isoflurane applied through a nose cone while on a heating pad to maintain body temperature at 37°C. Proparacaine hydrochloride 0.5% ophthalmic eye drops (Ursapharm Arzneimittel GmbH) were applied to numb the eyes, and subcutaneous injection of 5mg/kg Metacam alleviated pain (Meloxacam, Boehringer Ingelheim). The lateral canthus was de-vascularized by clamping with a hemostat (Fine Science Tools) for 10 seconds. Using a Leica dissection microscope, a lateral canthotomy allowed visualization of the posterior pole. While firmly holding the conjunctiva with a jeweler forceps, the conjunctiva was cut perpendicular to the posterior pole. The surrounding muscle was carefully dissected as to not puncture the vascular plexus. The optic nerve was pinched 1mm from the posterior pole for 4 seconds using a curved N7 self-closing forceps (Dumont). Triple antibiotic ointment was applied to the eye directly after the surgery to prevent infection.

For all surgical procedures, mice were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) and received meloxicam (1 mg/kg) as analgesia after the operation.
Sciatic nerve injury was achieved by either transecting or crushing the sciatic nerve. Briefly, the skin and muscle at the middle thigh level was dissected to expose the sciatic nerve. A transection was performed as a conditioning lesion. The nerves on the contralateral side were exposed, but no transection was performed, which acted as a sham control. The sciatic nerve crush injury was conducted using a hemostat (Fine Science Tools) for 30 s. For the CL group, a second crush injury was performed 3 d later proximal to the first crush site.
Dorsal column spinal cord crush was performed using a method similar to previously described methods. Briefly, an incision was made over the thoracic vertebrae, and a laminectomy was conducted to expose the T8 spinal cord. A T8 dorsal hemi-crush injury was performed using modified Dumont #5 forceps at a depth of 0.6 mm. Four weeks later, CTB488 was injected into the sciatic nerve to retrogradely trace the ascending sensory axon. Mice were killed 2 d later for examination. For the acute experiment, CTB was injected shortly after dorsal column crush, and mice were killed after 2 d.