Axoplasm Isolation from Rat Sciatic Nerve

Summary

We demonstrate a protocol for axoplasm isolation from adult rat sciatic nerve based on dissection of nerve fascicles and incubation in hypotonic medium to release myelin and lyse non-axonal structures, followed by extraction of the remaining axon-enriched material.

Abstract

Isolation of pure axonal cytoplasm (axoplasm) from peripheral nerve is crucial for biochemical studies of many biological processes. In this article, we demonstrate and describe a protocol for axoplasm isolation from adult rat sciatic nerve based on the following steps: (1) dissection of nerve fascicles and separation of connective tissue; (2) incubation of short segments of nerve fascicles in hypotonic medium to release myelin and lyse non-axonal structures; and (3) extraction of the remaining axon-enriched material. Proteomic and biochemical characterization of this preparation has confirmed a high degree of enrichment for axonal components.

Protocol

This protocol allows axoplasm isolation with minimization of glial and vascular tissue contamination. The method yields approximately 70-100 μg total axoplasm protein per one 8-10 week old Wistar rat. 1. Dissect Sciatic Nerves Euthanize two rats by CO2 inhalation followed by cervical dislocation. Confirm death by palpating for lack of heartbeat prior to beginning dissection. Swab the dissection area with 70% ethanol. Cut the skin, separate …

Discussion

Biochemical and proteomic analyses have confirmed that this procedure reduces serum and glial cell contamination ³ as compared to previously described methods for axoplasm isolation by mechanical squeeze ¹. We have used this axoplasm isolation protocol to explore dynein-based retrograde signaling after sciatic nerve injury², and expect it to have utility in the exploration of many other processes in adult peripheral nerve, including axon-glia interactions ⁴.

Materials

• Male Wistar rats (8-10 weeks old)
• PBS 0.2X and PBS 1X solutions containing protease inhibitors (Roshe) 2 tablets per 50 ml
• Eppendorfs for 1.5 ml
• Sterilized plastic dishes 35 mm
• Dissecting tools including: scissors and fine forceps
• Binocular and table light

Antibodies

Mouse anti-GFAP clone G-A-5 was from Sigma (G6171). Rabbit anti-Albumin was from Cedarlane (CLAG5140). Mouse anti-Tubulin β3 and rabbit anti-gERK were from Sigma (T2200 and M5670 respectively).