Zalewski A A, Fahy G M, Azzam N A, Azzam R N
Laboratory of Neural Control, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
J Comp Neurol. 1993 May 1;331(1):134-47. doi: 10.1002/cne.903310109.
Donor Schwann cells, perineurial cells, and vasculature are known to survive in grafts of peripheral nerve. In the present study, we attempted to cryopreserve nerve to determine whether these cellular components of nerve would survive after transplantation and support host axonal regeneration through the graft. Four-centimeter lengths of peroneal nerves were removed from inbred adult American Cancer Institute (ACI) rats and placed into vials that contained a cryoprotective mixture of dimethyl sulfoxide and formamide (DF) at room temperature. Each vial with nerves in DF was cooled at a rate of 1-1.5 degrees C/minute down to -40 degrees C at which point the vials were plunged into liquid nitrogen at -196 degrees C. After 5 weeks of storage, the nerves were thawed and DF removed. Some of the cryopreserved-thawed ACI nerves were transplanted as isografts into the legs of ACI rats. Other ACI nerves were used as allografts and inserted into immunologically normal Fischer (FR) rats that were untreated or were immunosuppressed with the drug Cyclosporin A (Cy-A). At surgery, only one end of the nerve graft was joined to the cut proximal end of the peroneal nerve of the host. The cellular elements of ACI grafts were present at 5 weeks in grafts removed from ACI rats and FR rats treated with Cy-A. Non-immunosuppressed FR rats rejected ACI nerves as did FR rats in whom Cy-A was stopped after 5 weeks of treatment. All surviving ACI grafts underwent Wallerian degeneration and consisted of columns of Schwann cells, which in their proximal portion were associated with regenerating host axons. The donor perineurial sheath and vasculature were also present in surviving grafts. ACI isografts only were examined 20 weeks postoperatively. All normal tissue components survived in these older grafts and contained regenerated and myelinated host axons throughout their 4 cm lengths. These results demonstrated that the cellular elements of nerve can be cryopreserved, and after transplantation, survive and function. Because nerves survived after prolonged cryopreservation, it seems feasible to establish a nerve bank from which grafts can be withdrawn to repair gaps in injured nerves. However, cryopreserved nerves used as allografts remain immunogenic and require immunosuppression for their survival.
已知供体雪旺细胞、神经束膜细胞和脉管系统可在周围神经移植物中存活。在本研究中，我们试图冷冻保存神经，以确定神经的这些细胞成分在移植后是否会存活，并支持宿主轴突通过移植物再生。从近交系成年美国癌症研究所（ACI）大鼠身上取出4厘米长的腓神经，在室温下放入含有二甲基亚砜和甲酰胺（DF）冷冻保护混合物的小瓶中。每个装有处于DF中的神经的小瓶以1-1.5℃/分钟的速率冷却至-40℃，此时将小瓶投入-196℃的液氮中。储存5周后，将神经解冻并去除DF。一些冷冻保存-解冻后的ACI神经作为同基因移植物移植到ACI大鼠的腿部。其他ACI神经用作异基因移植物，插入未经处理或用环孢素A（Cy-A）免疫抑制的免疫正常的Fischer（FR）大鼠体内。在手术时，仅将神经移植物的一端与宿主腓神经的切断近端相连。在从用Cy-A处理的ACI大鼠和FR大鼠身上取出的移植物中，5周时ACI移植物的细胞成分仍然存在。未免疫抑制的FR大鼠排斥ACI神经，在用Cy-A治疗5周后停药的FR大鼠也是如此。所有存活的ACI移植物均发生华勒氏变性，由雪旺细胞柱组成，其近端部分与再生的宿主轴突相关。存活的移植物中也存在供体神经束膜和脉管系统。仅对术后20周的ACI同基因移植物进行了检查。在这些较老的移植物中，所有正常组织成分均存活，并且在其4厘米长度内均含有再生和有髓鞘的宿主轴突。这些结果表明，神经的细胞成分可以冷冻保存，并且在移植后能够存活并发挥功能。由于神经在长时间冷冻保存后仍能存活，因此建立一个神经库似乎是可行的，从中可以取出移植物来修复受损神经中的间隙。然而，用作异基因移植物的冷冻保存神经仍然具有免疫原性，需要免疫抑制才能存活。
