Preserving peripheral nerves in desiccating conditions with a mixture of carbon dioxide and oxygen gases.

The surgical repair of peripheral nerve injuries associated with nerve deficits may be hindered by the dearth of suitable donor nerves (1, 2). Here, we report that donor rat peripheral nerve grafts were able to achieve effective reinnervation of targeted muscle groups after preservation in desiccating conditions with pressurized, mixed carbon dioxide and oxygen gases. Briefly, 20-mm–long sciatic nerve segments were harvested from Fischer rats and randomly divided into four groups: group A nerve segments were transplanted immediately without preservation, group B nerve segments were exposed to carbon dioxide and oxygen mixed gases (partial pressure of carbon dioxide, 100 hPa; oxygen, 900 hPa), group C nerve segments were immersed in University of Wisconsin solution, and group D nerve segments were exposed to pure oxygen (1000 hPa). The equipment (3, 4) (see Figure 1, Supplemental Digital Content 1, http://links.lww.com/TP/A264), maintaining the 1000 hPa pressure, was stored for 3 weeks at 4°C (detailed in the Methods, Supplemental Digital Content 2, http://links.lww.com/TP/A265). As a result, notable differences in the state of the stored nerve grafts were revealed by electron microscopy. The nerves in group A had a normal myelin sheath and normal Schwann cells, whereas those in group B showed some evidence of degenerative changes throughout the myelin sheath and Schwann cells and an intact basal lamina and cytoplasmic membrane. Group C showed clear evidence of demyelination and disruption of Schwann cells. Group D revealed complete destruction of the myelin sheath and no Schwann cells (Fig. 1A). Twelve weeks after the nerve transplant, during electrophysiologic study for nerve graft function, the values (on the experimental side was expressed as a percentage of that on the contralateral side) of the amplitude of compound muscle action, a parameter used to evaluate the activity of conduction in the preserved nerves of the gastrocnemius of the experimental limb, were (mean SD) 47.1 18.0, 39.9 16.3, 8.8 3.6, and 1.8 0.9 in groups A, B, C, and D (Fig. 1B). The compound muscle action potential amplitudes in group B were significantly greater than those in groups C and D (P 0.001). In histologic and morphologic studies, myelinated axons were dispersed individually in group B, as were those in group A (controls). However, groups C and D showed small, myelinated and nonmyelinated axons, grouped in fascicles that were bound by connective tissue (see Figure 2A, Supplemental Digital Content 3, http://links.lww.com/TP/A266). Moreover, the capability of regeneration of the nerve fibers in the graft segments was demonstrated by immunofluorescence staining. The green fasciculi were arranged regularly and densely in groups A and B and disorderly and sparsely in groups C and D (see Figure 2B, Supplemental Digital Content 3, http://links.lww.com/TP/A266). The mean myelinated fiber equivalent diameter (micrometer) of the grafts in groups A, B, C, and D were (mean SD) 3.03 1.47, 2.81 1.30, 1.67 0.73, and 1.49 0.70 (Fig. 1C). The mean myelinated fiber equivalent diameter of group B grafts, similar to that of group A, was significantly greater than that of groups C and D (P 0.048, P 0.024). The values (expressed as a percentage) of the musculus triceps surae weight were (mean SD) 67.0 5.0, 62.9 6.0, 55.2 5.1, and 54.8 4.3 in groups A, B, C, and D (see Figure 3A, Supplemental Digital Content 4, http://links.lww.com/TP/A267). The weights of group B subjects were significantly higher than those in group C (P 0.015) and group D subjects (P 0.008), and similar to those in group A subjects. A histologic study of the musculus triceps surae transverse sections showed that the muscle cells in groups A and B were slightly atrophied, and atrophied muscle cells were grouped in fascicles that were bound by connective tissue in groups C and D (Fig. 1D). The areas of the muscle fiber transverse section (expressed as a percentage) were (mean SD) 76.9 12.5, 69.2 11.6, 57.4 6.7, and 42.9 5.0 in groups A, B, C, and D (see Figure 3B, Supplemental Digital Content 4, http://links.lww.com/TP/A267). The group B values were significantly higher than those in group C (P 0.026) and group D (P 0.011), and similar to group A values. To the best of our knowledge, this is the first study to report that the donor peripheral nerves can be preserved successfully, easily, and relatively inexpensively in a desiccated state, without drugs or a preservation solution, and in conditions similar to the animal’s state of dormancy in nature. The grafted nerves regained their activity after long-term preservation, suggesting that carbon dioxide and desiccating conditions may have critical roles in tissue preservation. Fei Teng Wendong Xu Xiao-Kang Li Naoyuki Hatayama Kunihiro Seki Jianguang Xu 1 Department of Hand Surgery Huashan Hospital Fudan University Shanghai, China 2 Currently, Department of Emergency Beijing Chao-Yang Hospital Capital Medical University Chaoyang District, Beijing, China 3 State Key Laboratory of Medical Neuroscience, Fudan University Shanghai, China 4 Laboratory of Transplantation Immunology National Research Institute for Child Health and Development Setagaya-ku, Tokyo, Japan