Preservation of rat limbs by hyperbaric carbon monoxide and oxygen

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.

with organ preservation led us to consider whether the method might be effective for preserving amputated extremities.
We aimed to assess the efficacy of using CO and O 2 under high pressures to preserve rat limbs. To do so, we transplanted preserved rat hind limbs to syngeneic recipient rats in which the corresponding limbs had been removed.

Results
Limb survival rates after preservation under different conditions. Donor rat limbs were preserved in a chamber filled with CO and O 2 for 3 days (CO + O 2 3 days) or 7 days (CO + O 2 7 days) at 4 °C under high pressure. Positive and negative control groups were created by using non-preserved limbs (NP group) and limbs wrapped in SMG for 3 days at 4 °C (SMG 3 days), respectively.
At postoperative day (POD) 7, the survival rate of transplanted limbs was 100% in the NP group, but graft failure occurred in one of those limbs because of self-harm and subsequent infection at POD 17 (n = 8; Fig. 1). The survival rate of transplanted limbs was 100% in the CO + O 2 3 days group at POD 7, but graft failure occurred in two of the limbs at POD 14 (n = 14; Fig. 1). At POD 3, the survival rate of transplanted limbs was 100% in the CO + O 2 7 days group, but there was graft failure in five limbs by POD 14 (n = 10; Fig. 1). All limbs in the groups treated with CO + O 2 for 10 days, CO + N 2 for 3 days, O 2 + N 2 for 3 days, and SMG for 3 days (n = 6 each) underwent necrosis by POD 3, precluding further evaluation. Graft survival was determined by macroscopic diagnosis and bleeding from the foot tip after (27-gage) needlestick injury. Graft failure was assumed in transplanted limbs when there was color to change black or bleeding was not present.
Macroscopic and computed tomography images at POD 90. Macroscopically, there were no observable differences in the skin of the non-transplanted right hind limb and the transplanted left hind limb in the NP, CO + O 2 3 days, and CO + O 2 7 days groups ( Fig. 2A). Computed tomography showed slight atrophy of the triceps surae muscles of transplanted limbs in the NP, CO + O 2 3 days, and CO + O 2 7 days groups when compared with the intact limbs ( Fig. 2A).
Wet weights and histological images of the triceps surae muscles at POD 90. Wet muscle weight decreased significantly in descending order from the NP, CO + O 2 3 days, and CO + O 2 7 days groups, respectively, when compared with the Intact group (Fig. 2B). Atrophy of whole muscle fibers was observed in the CO + O 2 3 days and CO + O 2 7 days groups compared with the NP group but was more severe in the CO + O 2 7 days group than in the CO + O 2 3 days group (Fig. 2B).
Sciatic-tibial nerve conduction velocities at POD 90. The sciatic-tibial motor nerve conduction velocity did not change in any of the groups (Fig. 3Aa) but did decrease compared with the Intact group in the NP, CO + O 2 3 days, and CO + O 2 7 days groups (Fig. 3Ab). However, no significant differences were found among the NP, CO + O 2 3 days, and CO + O 2 7 days groups (Fig. 3Ab).
Walking appearance at POD 90. Walking appearance analysis showed that rats could walk using the transplanted limbs at POD 90 in the NP, CO + O 2 3 days, and CO + O 2 7 days groups (Fig. 3B). Foot stamps in the NP and the CO + O 2 3 days groups had almost the same appearances, but those in the CO + O 2 7 days group were obviously different.
Preserving limbs in hyperbaric gases after initial wrapping in SMG for 6 h. To assess the potential role of hyperbaric gases to preserve limbs in current clinical settings, we compared groups receiving limbs that were not preserved, that were preserved by wrapping in SMG at 4 °C group for 24 h, and that were preserved by wrapping in SMG for 6 h before being preserved in a hyperbaric chamber with CO and O 2 for 18 h at 4 °C throughout. Blood samples were then collected from recipients 1 h after transplantation and potassium and creatine phosphokinase levels were measured (Fig. 4B). The creatine phosphokinase but not potassium levels in the group that received CO and O 2 after initial SMG preservation were significantly lower than those only preserved with SMG.

Discussion
We verified the effect of hyperbaric CO and O 2 on amputated limb preservation in rats. Although muscle mass decreased, 86% of limbs preserved for 3 days and 50% of limbs preserved for 7 days survived more than 90 days after transplantation. These results indicate that using CO and O 2 at high pressure could greatly improve the preservation time of amputated limbs.
The blood flow of amputated rat limbs preserved for 3 and 7 days with hyperbaric CO and O 2 were maintained with a probability of 50% or more by macroscopic diagnosis and needlestick bleeding at POD 90. Neural evaluations of these limbs at POD 90 also indicated that the motor and sensory activities did not change in comparison with the transplanted limbs immediately after amputation. Because LEW/SsN Slc rats are an inbred strain, immune rejection was not taken into consideration in this experiment. However, muscle mass significantly decreased, and both severe muscle atrophy and fibrosis were observed in the limbs preserved for 7 days, with the rats ultimately unable to walk normally. Thus, the amputated limbs preserved for 3 and 7 days with hyperbaric CO and O 2 could be engrafted but with deteriorated function.
Recently, using histological and 18 F-fluorodeoxyglucose positron emission tomography analyses, we revealed that rat heart function was well preserved until 24 h, but not 48 h, after storage in hyperbaric CO and O 2 conditions 20 . Because heart function needs to be available immediately after transplantation, and because it is difficult to recover heart function, we recommended that hearts should not be preserved in hyperbaric CO and O 2 for more than 24 h. By contrast, human extremities can be rehabilitated after replantation, and in the case of digits, few muscles are involved. This led us to consider that preservation of amputated extremities might be feasible for 3 or 7 days with our method. And treatment is orderly performed for maintaining life and the priority of operation for the amputated extremities is postponed in the cases of multiple-trauma. Therefore, the IRI is unavoidable in the replantation and it should be important for the human amputated extremities to extend the preservable time by our method in hyperbaric CO and O 2 .
Although the preservation of human amputated extremities could be extended by our method of storage in hyperbaric CO and O 2 , this method would not be readily available at or near the site of an accident. Indeed, this preservation method is complicated and requires specialist, bulky equipment. A more practical clinical scenario might involve an initial period of cold ischemia with SMG during transport to a dedicated clinical facility at which the hyperbaric gas system could be available. In this study, we showed significantly lower creatine phosphokinase levels in the blood samples of recipients who received limbs wrapped in SMG for 6 h before preservation in the chamber with CO and O 2 gases for 18 h compared with limbs wrapped in SMG for 24 h. These results suggested the possibility for clinical applicability of the hyperbaric CO and O 2 preservation method.
In this study, we showed that hyperbaric CO and O 2 treatment can extend the time limit for preservation and reduce IRI. It has been reported that CO could influence signaling pathways via soluble guanylate cyclase and/or mitogen-activated protein kinase 13,14,21 and that the inhalation of CO has antiproliferative, antioxidant, anti-inflammatory, and antiapoptotic effects 13,14 . Previous reports also indicate that in transplantation of heart, lung, kidney, liver, and intestinal allografts or xenografts, tissues subject to CO had either reduced or prevented inflammation and apoptosis [22][23][24][25][26][27] . Hyperbaric O 2 treatment also reduces cell death by decreasing inflammatory and apoptosis signaling and by altering enzymatic antioxidant activity 17,28,29 . These results underpin the biological 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 O 2 for 18 h at 4 °C throughout. ***P < 0.001 by the Tukey-Kramer test.
SCIeNTIfIC REPORTs | (2018) 8:6627 | DOI:10.1038/s41598-018-25070-y plausibility of the effects hyperbaric CO and O 2 to reduce IRI in extracted organs. However, we only performed limb preservation under the same conditions we had previously used to preserve hearts 19,20 , yet it is known that the partial pressures of each gas and total pressure influence preservation. We therefore need to consider whether there is a more optimal ratio of the two gases and whether this varies by organ or limb.
Various approaches are currently available for the preservation of organs. Simple static cold storage is used primarily for heart and liver transplantation; hypothermic machine perfusion, for kidney transplantation; and the two layers method, for pancreatic transplantation 30 . Each preservation method has its own advantages and disadvantages making them appropriate to a given organ or situation. Traditionally, amputated limbs have been preserved by wrapping in SMG and placing on ice. There have been only a few reports on the development of long-term preservation methods for amputated limbs 31,32 . Preservation in hyperbaric CO and O 2 is a different approach, and we have provided the first evidence of successful replantation of amputated rat limbs preserved for up to 7 days. Therefore, we conclude that hyperbaric CO and O 2 therapy could be developed to preserve amputated limbs in the long term. Moreover, there have been many reports on the use of other medical gases for organ preservation, including the cell-protective effects of gases such as nitric oxide, hydrogen, hydrogen sulfide, xenon, and ozone 33 . Using these gases in hyperbaric chambers, either alone or in combination, might allow further expansion for "organ preservation". In conclusion, the preservation of amputated extremities in hyperbaric CO and O 2 could extend the time limits for replantation.

Methods
Animals. This experiment used an inbred line of LEW/SsN Slc rats (male, 10 weeks old, average weight: 230 g, range: 220-245 g, intact: n = 4, donors: n = 74, recipients: n = 56) that were purchased from the Shizuoka Laboratory Animal Center (Shizuoka, Japan). All handling and care of the rats conformed to the National Institutes of Health guidelines for animal research, and all experimental protocols involving animals were approved by the National Research Institute for Child Health and Development Animal Care and Use Committee (Permit Number: S-24059). All experiments involving animals were performed in accordance with the relevant guidelines and experimental protocols. All efforts were made to minimize animal suffering.
Experimental design. Amputated rat limbs were evaluated with orthotropic hind limb transplantation.
In the preservation chamber, a flask was placed with 50 mL of distilled water to maintain humidity. Rat limbs used immediately after extraction (i.e., NP group) served as the positive controls (n = 8), whereas limbs wrapped in SMG for 3 days at 4 °C (i.e., SMG 3 days group) served as negative controls (n = 6). Each limb underwent orthotopic transplant to syngeneic recipient rats and was evaluated by comparison with intact rat limbs (Intact: n = 4). Alteration in transplanted limb color to black indicated graft failure, and rats were then sacrificed by cardiac incision under deep anesthesia. At the end of the experimental time, triceps surae muscles of a sample were excised. At POD 90, transplanted limbs were evaluated for the following: macroscopic diagnosis, survival rate, computed tomography, wet weights, histology, triceps surae muscle motor and sensory nerve conduction tests, and walking track analysis.
We performed further study to assess the role of hyperbaric preservation in clinical medicine (Fig. 4A). For this, we compared groups receiving limbs that were not preserved (NP group: n = 6), that were preserved by wrapping in SMG for 24 h at 4 °C (SMG group: n = 6), and that were preserved by wrapping in SMG for 6 h before being preserved in a hyperbaric chamber with CO and O 2 (7000 hPa; PCO = 4000 hPa, PO 2 = 3000 hPa) for 18 h at 4 °C (SMG CO + O 2 group: n = 6) throughout. Blood samples were then collected from recipients 1 h after transplantation, and potassium and creatine phosphokinase levels were measured.
Surgical procedure. Preparation of donor and recipient limbs was performed while rats were deeply anesthetized with isoflurane (ISOFLU ® , Dainippon Sumitomo Pharma Co., Ltd.), using an inhalation anesthesia apparatus (Univentor 400 Anesthesia Unit, Univentor Ltd.). Anesthetic induction was performed in cages with a 4-5% concentration of isoflurane at a high flow rate (450-500 mL/min) for about 5 min. We then delivered an isoflurane concentration of 1.5-2.0% at 150-200 mL/min through a mask covering the rats' noses. Donor rats were administered heparin (50 U; Ajinomoto, Tokyo, Japan) by tail vein injection as pretreatment. The skin, muscles, vessels, nerves, and bone were cut around the circumference of the left hind limb near the level of the inguinal ligament. Blood was then removed from the femoral artery and replaced with Krebs-Henseleit solution at 4 °C. In recipient rats, femur intramedullary fracture fixation was stabilized by an 18-gage needle, and the muscles were sutured with 4-0 Vicryl. The femoral artery and vein were anastomosed with 10-0 nylon, and the sciatic nerve was sutured with 10-0 nylon epineural sutures under a surgical microscope. Blood flow was checked after 15 min, and the skin was closed. An injection of the analgesic Rimadyl (carprofen 5%; Pfizer, UK) was administered subcutaneously (0.1 mL/kg) until POD 7, and the condition of transplanted limbs was observed daily.
Computed tomography. Computed tomography analyses were performed with 0.65 mm slices using an Optima CT660 (GE Healthcare, Milwaukee, WI, USA) equipped with an X-ray tube working at 120 kV/100 mA.
Motor and sensory nerve conduction tests. All electrophysiological measurements were taken from the left hind limbs. Electrophysiological measurements were recorded using an electromyography/evoked potential measuring system (Neuropack S1 MEB-9400, Nihon Kohden Corp., Tokyo, Japan). Supramaximal stimuli were applied for all studies, and the velocities were calculated from the onset latency of the recorded potential. All sensory studies were digitally averaged until a clear waveform was obtained. Motor nerve conduction tests were performed by placing cathode needle electrodes in two positions (the sciatic notch and approximately 3 cm distally). The recording needle electrode was placed in the foot. Orthodromic sensory studies were performed using the motor recording electrode, described as the stimulating electrode above, with recording performed at the sciatic notch.
Walking track analysis. Walking track analysis was performed to examine replanted hind limb at POD 90.
Briefly, rats were allowed conditioning trials in a walking track (8.2 × 42 cm) darkened at one end. White office paper cut to the appropriate dimensions was placed on the bottom of the track. The rat's hind limbs were dipped in Chinese ink, and the rat was permitted to walk down the track, leaving its hind footprints on the paper. In addition, walking appearance was recorded with a digital camera (COOLPIX S8200; Nikon Corporation, Tokyo, Japan).

Light microscopy.
Hind limbs from each group were fixed in Bouin's solution for 3 h. The samples were then washed, dehydrated with an ethanol series, and embedded in paraffin. Serial 6 μm sections were cut with a microtome and stained with Gill's hematoxylin III and 2% eosin Y.
Serum biochemistry. For the serum biochemical evaluation the following levels were determined: potassium (K) and creatine phosphokinase (CPK).
Statistical analysis. Data are presented as means ± standard deviations, and significance differences were determined by analysis of variance with post-hoc tests. A p-value < 0.05 was considered statistically significant.
Data availability. All data generated during this study are included in this published article.