Optimal freezing and thawing for the survival of periphheral nerves in severed rabbit limbs

Zexing Zhu, Lin Qiao, Yandong Zhao, Shuming Zhang
2014 Int J Clin Exp Pathol   unpublished
nism of cryopreservation is that low temperature can significantly slow down or even stop the enzymatic reactions in cells, leading to decreased cell metabolism [2]. At the temperature of liquid nitrogen of-196°C, all the intracel-lular biochemical reactions are ceased, and the cell metabolism is stopped. The cryopreserved cells at this temperature will not have neither apoptosis nor necrosis, and maintain their intact biological structures. However, the freezing and thawing processes can
more » ... processes can damage the cryopreserved cells and tissues. During slow freezing, water nucleation occurs first in the extracellular fluid, which then decreases the volume of extracellular fluid. The decreased extracellular fluid disrupts the osmo-larity balance between the intracellular and extracellular environments. The intracellular water then transfers to the extracellular environment , increasing the intracellular osmolarity and damaging the organelles. In the contrary, fast freezing induces water nucleation simultaneously in both the intracellular and extracellu-Original Article Abstract: This study aimed to investigate the optimal freezing and thawing procedures for the survival of peripheral nerves in severed rabbit limbs. Twenty New Zealand White rabbits were randomized into four groups: normal control, slow-freezing fast-thawing, slow-freezing slow-thawing, fast-freezing fast-thawing, with five animals in each group. The hind limbs of the rabbits were severed at 1 cm above the knee joint. The severed limbs were cryopreserved with various freezing and thawing procedures. The sciatic nerves were harvested and trypsinized into single nerve fibers for morphological evaluation. The cell viability of the nerve fibers was examined by staining with Calcein-AM and propidium iodide. The fluorescent intensity of the nerve fibers was measured with a laser scanning confocal microscope. The morphology of the nerve fibers in the slow-freezing fast-thawing group was very similar with that of the normal control group, with only mild demyelination. The slow-freezing fast-thawing group and slow-freezing slow-thawing group showed severely damaged nerve fibers. The fluorescent intensities of the nerve fibers was significantly different among the four groups, with a decreasing order of normal control, slow-freezing fast-thawing, slow-freezing slow-thawing, and fast-freezing fast-thawing (P < 0.05). Of the various cryopreservative procedures, slow-freezing fast thawing has the minimal effects on the survival of nerve fibers in severed rabbit limbs.