EFFECT OF DIFFERENT TEMPERATURES ON SYSTEM OF IN VITRO PHYSIOLOGICAL ENVIRONMENT FOSTERING LIMBS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

YIN Yefeng ¹ , WANG Jiangning ^1,2 , GAO Lei ² , LIU Ning ¹ , XIA Xiaoyong ² , QIN Xinyuan ² , ZUO Youwei ²

1Graduate School, Dalian University, Dalian Liaoning, 116622, P.R.China;;
2Department of Plastic and Reconstructive Surgery, Beijing Luhe Hospital of Capital Medical University. Corresponding author: WANG Jiangning, E-mail: dlwangjn@vip.sina.com;

Corresponding author：

Keywords：

In vitro foster care of amputated limb; Cryopreservation; Pig

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Objective To investigate the effects of different temperatures on the system of in vitro physiological environment fostering limbs. Methods Twenty-four limbs were harvested from 6 adult Bama mini pigs and were randomly divided into 4 groups (n=6) according to different temperatures: limbs were placed in in vitro physiological environment foster-ing limbs at 26℃ (group A), 4℃ (group B), 10℃ (group C), and 18℃(group D). After 12 hours of perfusion, the morphology observation was done for the structure and ultrastructure changes of the skeletal muscle by light microscope and transmission electron microscope. The mRNA levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) were detected by real-time fluorescent quantitative PCR (RT-qPCR). Results Histological results showed that the skeletal muscle exhibited mild edema, integrity of the sarcolemma, and occasional perivascular inflammatory cell infiltration in groups B, C, and D, meanwhile, the cells of group C had normal morphology; however, muscle fibers degenerated, muscle cells were seriously damaged, a great number of inflammatory cells infiltrated in the fractured muscle fibers in group A. Transmission electron microscope results showed as follows: the muscle fibers arranged in disorder, and many focal solubility necrosis occurred in group A; the muscle fibers arranged in order relatively and sarcolemma was still intact, with mild swelling and flocculent degenerative mitochondria in group B; a large number of muscle fibers arranged in order and regularity with clear sarcomere in group C; and the muscle fibers arranged in disorder and irregularity and partly dissolved in group D. RT-qPCR results showed that the expressions of inflammatory factor TNF-α and IL-1β mRNA in group A were significantly higher than those in groups B, C, and D (P lt; 0.05); the expressions were significantly lower in groups B and C than in group D, and in group C than in group B (P lt; 0.05). Conclusion In the system of in vitro physiological environment fostering limbs, temperature plays an important role in the preservation of amputated limbs. It is suggested that 10℃ can significantly attenuate the reperfusion-induced skeletal muscle cell injuries in this system.

Citation： YIN Yefeng,WANG Jiangning,GAO Lei,LIU Ning,XIA Xiaoyong,QIN Xinyuan,ZUO Youwei. EFFECT OF DIFFERENT TEMPERATURES ON SYSTEM OF IN VITRO PHYSIOLOGICAL ENVIRONMENT FOSTERING LIMBS. Chinese Journal of Reparative and Reconstructive Surgery, 2013, 27(1): 72-76. doi: Copy

1.	张智, 蒋祖言, 王爱民. 断肢保存的研究进展. 中国修复重建外科杂志, 2000, 14(3): 189-192.
2.	Wilson YT, Lepore DA, Riccio M, et al. Mild hypothermia protects against ischemia reperfusion injury in rabbit skeletal muscle. Br J Plast Surg, 1997, 50(5): 343-348.
3.	Mowlavi A, Neumeister MW, Wilhelmi BJ, et al. Local hypothermia during early reperfusion protects skeletal muscle from ischemia reperfusion injury. Plast Reconstr Surg, 2003, 111(1): 242-250.
4.	Wagh M, Pantazi G, Romeo R, et al. Cold storage of rat skeletal muscle free flaps and pre-ischemic perfusion with modified UW solution. Microsurgery, 2000, 20(7): 343-349.
5.	Tsuchida T, Kato T, Yamaga M, et al. Effect of perfusion during ischemia on skeletal muscle. J Surg Res, 2001, 101(2): 238-241.
6.	Yeh LS, Gregory CR, Theriault BR, et al. A functional model for whole limb transplant at ion in the rat. Plast Reconstr Surg, 2000, 105(5): 1704-1711.
7.	王江宁, 尹叶锋, 高磊, 等. 不同氧体积分数气体对体外模拟体内生理环境寄养断肢系统血氧分压的影响. 中国组织工程研究, 2012, 16(5): 855-858.
8.	Wang JN, Wang SY, Wang ZJ, et al. Temporary ectopic implantation for salvage of amputated lower extremities: case reports. Microsurgery, 2005, 25(5): 385-389.
9.	St Peter SD, Imber CJ, Friend PJ. Liver and kidney preservation by perfusion. Lancet, 2002, 359(9306): 604-613.
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11.	崔志民, 谢晞衷, 孟宏, 等. 创伤性四肢血管损伤修复经验及教训. 中国修复重建外科杂志, 1995, 9(1): 42.
12.	韦正超, 金文涛, 张峻峰. 四肢血管损伤的手术治疗. 中国修复重建外科杂志, 2002, 16(3): 191-192.
13.	Wang JN, Tong ZH, Zhang TH, et al. Salvage of amputated upper extremities with temporary ectopic implantation followed by replantation at a second stage. J Reconstr Microsurg, 2006, 22(1): 15-20.
14.	王江宁, 童致虹, 张铁慧, 等. 暂时性异位断足寄养再回植术. 中国修复重建外科杂志, 2003, 17(1): 46-49.
15.	李靖年, 李邰, 赵文志, 等. 局部低温对肢体缺血/再灌注损伤的保护作用实验研究. 中国矫形外科杂志, 2000, 7(1): 53-55.
16.	Awerbuck D, Luong V, Plyley MJ, et al. Skeletal muscle form and function after 4hr ischemia-hypothermia. J Surg Res, 1994, 57(4): 480-486.
17.	Toyoda T, Suzuki S, Kassell NF, et al. Intraischemic hypothermia attenuates neutrophil infiltration in the rat neocortex after focal ischemia-reperfusion injury. Neurosurgery, 1996, 39(6): 1200-1205.
18.	吴桂昌, 陆雪芬, 徐恩. 亚低温对大鼠脑缺血再灌注后炎症反应的作用研究．中风与神经疾病杂志, 2004, 21(5): 434-436.

1. 张智, 蒋祖言, 王爱民. 断肢保存的研究进展. 中国修复重建外科杂志, 2000, 14(3): 189-192.
2. Wilson YT, Lepore DA, Riccio M, et al. Mild hypothermia protects against ischemia reperfusion injury in rabbit skeletal muscle. Br J Plast Surg, 1997, 50(5): 343-348.
3. Mowlavi A, Neumeister MW, Wilhelmi BJ, et al. Local hypothermia during early reperfusion protects skeletal muscle from ischemia reperfusion injury. Plast Reconstr Surg, 2003, 111(1): 242-250.
4. Wagh M, Pantazi G, Romeo R, et al. Cold storage of rat skeletal muscle free flaps and pre-ischemic perfusion with modified UW solution. Microsurgery, 2000, 20(7): 343-349.
5. Tsuchida T, Kato T, Yamaga M, et al. Effect of perfusion during ischemia on skeletal muscle. J Surg Res, 2001, 101(2): 238-241.
6. Yeh LS, Gregory CR, Theriault BR, et al. A functional model for whole limb transplant at ion in the rat. Plast Reconstr Surg, 2000, 105(5): 1704-1711.
7. 王江宁, 尹叶锋, 高磊, 等. 不同氧体积分数气体对体外模拟体内生理环境寄养断肢系统血氧分压的影响. 中国组织工程研究, 2012, 16(5): 855-858.
8. Wang JN, Wang SY, Wang ZJ, et al. Temporary ectopic implantation for salvage of amputated lower extremities: case reports. Microsurgery, 2005, 25(5): 385-389.
9. St Peter SD, Imber CJ, Friend PJ. Liver and kidney preservation by perfusion. Lancet, 2002, 359(9306): 604-613.
10. 侯忠军. 加氧静脉血间断灌注延长离断肢体存活时限的实验研究. 佳木斯: 佳木斯大学, 2002.
11. 崔志民, 谢晞衷, 孟宏, 等. 创伤性四肢血管损伤修复经验及教训. 中国修复重建外科杂志, 1995, 9(1): 42.
12. 韦正超, 金文涛, 张峻峰. 四肢血管损伤的手术治疗. 中国修复重建外科杂志, 2002, 16(3): 191-192.
13. Wang JN, Tong ZH, Zhang TH, et al. Salvage of amputated upper extremities with temporary ectopic implantation followed by replantation at a second stage. J Reconstr Microsurg, 2006, 22(1): 15-20.
14. 王江宁, 童致虹, 张铁慧, 等. 暂时性异位断足寄养再回植术. 中国修复重建外科杂志, 2003, 17(1): 46-49.
15. 李靖年, 李邰, 赵文志, 等. 局部低温对肢体缺血/再灌注损伤的保护作用实验研究. 中国矫形外科杂志, 2000, 7(1): 53-55.
16. Awerbuck D, Luong V, Plyley MJ, et al. Skeletal muscle form and function after 4hr ischemia-hypothermia. J Surg Res, 1994, 57(4): 480-486.
17. Toyoda T, Suzuki S, Kassell NF, et al. Intraischemic hypothermia attenuates neutrophil infiltration in the rat neocortex after focal ischemia-reperfusion injury. Neurosurgery, 1996, 39(6): 1200-1205.
18. 吴桂昌, 陆雪芬, 徐恩. 亚低温对大鼠脑缺血再灌注后炎症反应的作用研究．中风与神经疾病杂志, 2004, 21(5): 434-436.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF DIFFERENT TEMPERATURES ON SYSTEM OF IN VITRO PHYSIOLOGICAL ENVIRONMENT FOSTERING LIMBS

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