Partial enzyme digestion facilitates regeneration of crushed nerve in rat

Abstract

Peripheral nerve injury is a life-changing disability with significant socioeconomic consequences. In this rat model, we propose that partial enzyme digestion can facilitate the functional recovery of a crushed nerve. The sciatic nerves were harvested and in vitro cultured with the addition of Liberase to determine the appropriate enzyme amount in the hyaluronic acid (HA) membrane. Then, the sciatic nerve of adult male Sprague-Dawley rats was exposed, crushed, and then treated with partial enzyme digestion (either 0.001 or 0.002 unit/mm² Liberase-HA membrane). The sciatic function index (SFI) for functional recovery of the sciatic nerve was evaluated. After 2 h of in vitro digestion, fascicles and axons were separated from each other, with the cells mobilized. Greater destruction of histology structures occurred in the high enzyme (Liberase-HA membrane at 0.002 unit/mm²) group at 24 h than in the low enzyme (0.001 unit/mm²) group at 48 h. In the SFI evaluation, the improvement in 0.001 unit/mm² Liberase group was significantly better than control and 0.002 unit/mm² Liberase group. Our study demonstrated that appropriate enzyme digestion had a significantly faster and earlier recovery.

Keywords: Liberase; nerve regeneration; partial enzyme digestion; rat model.

Figure 1

The flow-chart of experimental design.

Figure 2

The histological evaluation of sciatic nerve grafts after digestion. H/E stain showed that, with 2 h of digestion, the nerve bundle structure became loose, and fascicles and axons were separated from each other (scale bar = 100 µm).

Figure 3

The microscopic morphology of the sciatic nerve grafts during the digestion process. At 0 h of enzyme digestion, the nerve endings were compact without individual axon visible (a and b). After 4 h after enzyme digestion, nerve ends became more loosened. Individual nerve axon can be observed (c and d); the axon with its attached SCs can be identified at higher magnification (e). After 30 h of digestion, cells are mobilized from their original histology structures. Exposed axon (yellow-circled) and cells mobilized from axon, possible SCs (red-arrowed), can be seen (f–h) (scale bar = 100 µm).

Figure 4

The quantitative and microscopic evaluation of cells released from sciatic nerve grafts after digestion. (a) Cell number and cell viability of mobilized SCs by different liberase concentration media. The total number of retrieved cells from different liberase concentrations differed significantly (P < 0.005). In comparison with the 1X liberase-treatment group (0.74 unit/mL Liberase: 1.83 ± 0.05 × 10⁵ cells), it was higher in the 2X liberase-treatment group (1.48 unit/mL Liberase: 1.94 ± 0.06 × 10⁵ cells). However, this difference in all treatment groups was not significant (P > 0.2) in the cell counts of viable cells according to their neutral red stain. Influence of the concentration of liberase-treatment group on the viability of cells was even more obvious when it presented as the ratio of cells stained with neutral red; the viability of 2X liberase-treatment group (76.3 ± 2.3%) was significantly lower than that of 2X liberase-treatment group (82.2 ± 2.1%) (P < 0.005). (N = 9; *: P < 0.05, **: P < 0.005) (N = 9; *: P < 0.05, **: P < 0.005). (b) The microscopic evaluation of cells released from sciatic nerve grafts after digestion. There were some smaller and densely packed cells visible before cold irrigation (a); after cold irrigation, those cells weakly adhered to dishes were collected (red-arrowed) (b). Positive cytoplasmic staining (green) of S-100 demonstrated the viable SCs; while other cells showed positive DAPI nuclear staining (blue) in the background (c and d) (scale bar in A & B = 100 µm).

Figure 5

The effect of different Liberase concentrations. Liberase enzyme loosened the gross structure of the nerve graft by digested collagens. The results showed that more collagen was digested by the Liberase-HA membrane at 0.002 unit/mm² for 24 h (c and g) than at 0.001 unit/mm² for 48 h (d and h). (a–d) H/E stains; (e–h) type IV collagen stain. (b and f) Liberase-HA membrane at 0.001 unit/mm², 24 -h digestion; (c and g) Liberase-HA membrane at 0.002 unit/mm², 24 -h digestion; (d and h): Liberase-HA membrane at 0.001 unit/mm², 48 -h digestion (200×) (scale bar = 200 µm).

Figure 6

Digital analysis of the type IV collagen. After 24-hour digestion, type IV collagen amount of the 1X enzyme (Liberase-HA membrane at 0.001 unit/mm²) group was 13.9% less than that of the control group. While for the 2X enzyme (0.002 unit/mm²) group, it was 34.2% less than that of the control group. In addition, the 2X enzyme group digested statistically more collagen in 24 h than the 1X enzyme (0.001 unit/mm²) in 48 h.

Figure 7

The in vivo effect of partial enzyme digestion on crushed nerve regeneration. (a) The in vivo effect of different Liberase concentrations. The improvement in enzyme 1X group (Liberase at 0.74 unit/mL) was significantly better at 3, 4, 5, 6, and 11 weeks after the injury (p < 0.05) than the control and enzyme 2X group (1.48 unit/mL). The functional results of the control group were significantly better than those of the enzyme 2X group throughout the whole test period. (b) Liberase-HA Membrane Facilitated SFI Recovery. There had been a significantly better improvement for enzyme digestion since 3 weeks after the injury. However, after 7th week, there was no recovery difference between control and enzyme digestion group, except at the 11th and 13th weeks. (c) Cumulative Data for Effective Liberase-HA Membrane on SFI Recovery. There had been a significantly better improvement for 1X enzyme digestion than that of control since 3 weeks after the injury; however, there was no recovery difference between control and enzyme digestion group after the 12th week. While the effect of 2X enzyme digestion seems inferior to that of control and 1X enzyme digestion group (control group: n = 10 before 12th week; n = 5 at 13th–16th week; 1X enzyme digestion group: n = 9 before 12th week; n = 5 at 13th–16th week; 2X enzyme digestion group: n = 5 before 12th week; n = 0 at 13th–16th week).