Preservation of rat limbs by hyperbaric carbon monoxide and oxygen

Abstract

Cold ischemia times ranging from <6 h to as long as 24 h are generally quoted as the limits for attempting the replantation of amputated extremities. In this study, we aimed to assess the effect of hyperbaric carbon monoxide (CO) and oxygen (O₂) on rat limb preservation. Donor rat limbs were preserved in a chamber filled with hyperbaric CO and O₂ for 3 days (CO + O₂ 3 days) or 7 days (CO + O₂ 7 days). Positive and negative control groups were created by using non-preserved limbs (NP) and limbs wrapped in saline-moistened gauze for 3 days (SMG 3 days), respectively. The survival rate of transplanted limbs at postoperative day 90 was 88% in the NP and 86% in the CO + O₂ 3 days. The corresponding survival rate was 50% in the CO + O₂ 7 days at postoperative day 90 but was 0% in the SMG 3 days at postoperative day 3. Muscle mass decreased in the CO + O₂ 3 days and CO + O₂ 7 days compared with the NP, but sciatic-tibial nerve conduction velocities did not differ. These results indicate that amputated extremities preservation with hyperbaric CO and O₂ could extend the time limits of preservation, maintaining their viability for replantation.

Figure 1

Survival rate after transplantation by preservation method. Limbs were preserved in chambers filled with CO + O₂ for 3, 7, and 10 days, and CO + N₂, or N₂ + O₂ for 3 days under high pressure. Negative control was achieved by wrapping limbs in saline-moistened gauze (SMG) for 3 days at 4 °C. For the positive control, non-preserved (NP) limbs were used from donor rats.

Figure 2

Muscle evaluation in transplanted limbs at postoperative day 90. (A) Microscopic views (a–d), computed tomography images (e–h), and histological images (i–l). Intact = a, e, and i; non-preserved (NP) limbs = b, f, and j; 3 days of CO + O₂ = c, g, and k; and 7 days of CO + O₂ = d, h, and l. Circulation is normal in the transplanted left limbs in a, b, c, and d. Computed tomography images are shown above the green lines in the X-ray images from among each group. L, left hind limb. LT, left transplanted hind limb. White bar = 2 cm; Black bar = 100 μm. (B) Wet weights of triceps surae muscles. Data show the percentages of wet muscle weights in the transplanted left hind limbs compared with those in the non-transplanted right hind limbs per group. All data are expressed as means ± standard deviations, and analysis of variance (ANOVA) with the post-hoc Tukey–Kramer for statistical analysis. *P < 0.05 and **P < 0.01 by the Tukey–Kramer test.

Figure 3

Neural activity in transplanted limbs at postoperative day 90. (A) Sciatic–tibial nerve conduction velocities. Motor nerve conduction velocities (a) and sensory nerve conduction velocities (b). Data are shown per group and expressed as means ± standard deviations. The analysis of variance (ANOVA) with the Tukey–Kramer test post-hoc test was employed for statistical analysis. ***P < 0.001 by the Tukey–Kramer test. (B) Walking appearance. The black ink shows the representative walking track of each rat.

Figure 4

Evaluation of transplanted limb damage per group. (A) Experimental design. (B) The potassium (K) and creatine phosphokinase (CPK) levels in blood samples taken from recipients after transplantation. We compared groups receiving limbs that were not preserved (NP), preserved by wrapping in saline-moistened gauze (SMG) at 4 °C group for 24 h, and preserved by wrapping in SMG for 6 h before being preserved in a hyperbaric chamber with CO and O₂ for 18 h at 4 °C throughout. ***P < 0.001 by the Tukey–Kramer test.

Figure 5

Method of limb preservation in a hyperbaric chamber. (A) A schematic of the preservation method. The chamber was filled with CO at 4000 hPa and PO₂ at 3000 hPa. During limb preservation, a flask with 50 mL of distilled water (DW) was placed in the chamber to maintain humidity. (B) Experimental design.