Effect of prolonged ischaemic time on muscular atrophy and regenerating nerve fibres in transplantation of the rat hind limb

Abstract

Our aim was to test the influence of cold ischaemia on replanted limbs, focusing on muscular atrophy and neurological recovery. Inbred wild-type and green fluorescent protein (GFP) transgenic (Tg) Lewis rats aged 8-10 weeks were used. The amputated limbs were transplanted at several cold ischaemic times (0, 1, 8, 12, 24, 48, and 72 hours). An arterial ischaemic model and a denervation model were used as controls. To study nerve regeneration, a GFP limb was transplanted on to the syngenic wild Lewis rat. These animals were evaluated histologically, electrophysiologically, and immunohistochemically. The longer the ischaemic time, the more evident was atrophy of the muscles. Electrophysiological investigation showed that the latency at 3 weeks was longer in the transplantation models than in the normal controls, particularly in the longer ischaemia group. Larger numbers of migrating Schwann cells were seen in the group with no delay than in the group that had been preserved for 12 hours. Ischaemia after amputation of a limb causes muscle cells to necrose and atrophy, and these changes worsen in proportion to the ischaemic preservation time. A delay in nerve regeneration and incomplete paralysis caused by malregeneration also affect muscular atrophy.

Figure 1.

Gastrocnemius muscle specimens of rat hind limb. Haematoxylin and eosin stain; left, above: cross-section (original magnification ×40) left, below: longitudinal section (original magnification ×40), right above; cross section (original magnification ×400), right below: longitudinal section (original magnification ×400). Group A: transplantation model, preservation time (a) 1 hour, no distinguishable change, (b) at 72 hours, focal necrosis and degenerative muscle were seen. (c) Group B: arterial ischaemic model, no definite changes. (d) Group C: GFP transplantation model, uniform muscle atrophy was obvious. Scale bars, 50 μm (original magnification ×40), 5 μm (original magnification ×400).

Figure 2.

The results of measurement of the diameters of muscle cells in groups A, B, and C. In group A, the longer the ischaemic time, the more evident was muscle atrophy. In group B muscle atrophy was not pronounced. Group C showed severe atrophy. *p < 0.05.

Figure 3.

Distal motor latencies of the sciatic nerve. Comparison of shorter ischaemia group (0, 1 hours ischaemia), longer ischaemia group (8, 12 hours ischaemia), arterial ischaemic group, and normal controls. *p < 0.05.

Figure 4.

The GFP stained cells of the sciatic nerve in group D, the GFP transplantation model, at the point of 5 mm distal from suture. Cold preservation time 0 hour, at two weeks after transplantation. 489-nm wave length excitation light (original magnification ×40).