Schwann cell metabolic activity in various short-term holding conditions: implications for improved nerve graft viability

Abstract

Strategies for improvement of nerve regeneration and optimal conditions to prevent Schwann cell (SC) loss within a nerve transplant procedure are critical. The purpose of this study was to examine SC viability, which plays an important role in peripheral nerve regeneration, under various incubation conditions up to three hours. To address this issue, Schwann cell metabolic activity was determined using different independent test methods. The following experimental conditions were compared: SCs prepared from nerves were incubated in (1) isotonic saline solution (2) Dulbecco's modified Eagles medium as used for cell culturing, (3) Hannover bioreactor medium, and (4) Leibovitz's medium. SC metabolic activity of excised rat sciatic nerve was determined at 4°C, 18°C, and 37°C over 3 hrs. The results indicate that SC activity was optimized by the usage of Leibovitz's medium or HBRM at 37°C. Greater SC viability at the time of surgical nerve grafting could contribute to improved axonal regeneration and remyelination after nerve transplantation, and thus more successful functional recovery.

Figure 1

Conditions for testing the metabolic activity of Schwann cells in vitro after incubation in selected media including saline, DMEM with 10% FCS, HBRM, and Leibovitz's medium. Sciatic nerves were incubated in these selected media at 4°C, a room temperature 18°C and 37°C. For each temperature condition, Schwann cell metabolism was determined after 1 hrs, 2 hrs, and 3 hrs.

Figure 2

This Figure and respectively, Figure 3: Schwann cell metabolism indicated as fluorescence activity at 4°C (a), room temperature (b), and at 37°C (in (c)) incubated in the described media. Cell viability was measured by reduction of resazurin to resofurin by CellTiter-Blue (CTB) Cell Viability Assay. Most reduced cell viability was observed after 3-hour incubation of Schwann cells in DMEM at 4°C. Optimal cell viability with increase of Schwann cell metabolism can be seen after incubation in HBRM at 37°C. P < 0.05.

Figure 3

Figure 4

Schwann cell metabolism indicated as absorbance measured with MTS at 4°C (a), room temperature (b), and at 37°C (c) incubated in the described media. At 4°C all conditions resulted after 3 hours in massive reduction of cell viability. At room temperature incubation in HRBM and DMEM showed slightly better results than NaCl and Leibovitz's. Optimal cell viability with increase of Schwann cell metabolism can be seen after incubation in HBRM at 37°C as observed by independent determination of cell viability by CTB in Figure 2. P < 0.05.