Do Liquid Nitrogen-treated Tumor-bearing Nerve Grafts Have the Capacity to Regenerate, and Do They Pose a Risk of Local Recurrence? A Study in Rats

Abstract

Background: Under most circumstances, the resection of soft tissue sarcomas of the extremities can be limb-sparing, function-preserving oncologic resections with adequate margins. However, en bloc resection may require resection of the major peripheral nerves, causing poor function in the extremities. Although liquid nitrogen treatment has been used to sterilize malignant bone tumors, its use in the preparation of nerve grafts has, to our knowledge, not been reported. Hence, this study aimed to investigate the tumor recurrence and function after peripheral nerve reconstruction using liquid nitrogen-treated tumor-bearing nerves in a rat model.

Questions/purposes: (1) Do liquid nitrogen-treated frozen autografts have regeneration capabilities? (2) Do liquid nitrogen-treated tumor-bearing nerves cause any local recurrences in vivo in a rat model?

Methods: Experiment 1: Twelve-week-old female Wistar rats, each weighing 250 g to 300 g, were used. A 10-mm-long section of the right sciatic nerve was excised; the prepared nerve grafts were bridge-grafted through end-to-end suturing. The rats were grouped as follows: an autograft group, which underwent placement of a resected sciatic nerve after it was sutured in the reverse orientation, and a frozen autograft group, which underwent bridging of the nerve gap using a frozen autograft. The autograft was frozen in liquid nitrogen, thawed at room temperature, and then thawed in distilled water before application. The third group was a resection group in which the nerve gap was not reconstructed. Twenty-four rats were included in each group, and six rats per group were evaluated at 4, 12, 24, and 48 weeks postoperatively. To assess nerve regeneration after reconstruction using the frozen nerve graft in the nontumor rat model, we evaluated the sciatic functional index, tibialis anterior muscle wet weight ratio, electrophysiologic parameters (amplitude and latency), muscle fiber size (determined with Masson trichrome staining), lower limb muscle volume, and immunohistochemical findings (though neurofilament staining and S100 protein produced solely and uniformly by Schwann cells associated with axons). Lower limb muscle volume was calculated via CT before surgery (0 weeks) and at 4, 8, 12, 16, 20, 24, 32, 40, and 48 weeks after surgery. Experiment 2: Ten-week-old female nude rats (F344/NJcl-rnu/rnu rats), each weighing 100 g to 150 g, were injected with HT1080 (human fibrosarcoma) cells near the bilateral sciatic nerves. Two weeks after injection, the tumor grew to a 10-mm-diameter mass involving the sciatic nerves. Subsequently, the tumor was resected with the sciatic nerves, and tumor-bearing sciatic nerves were obtained. After liquid nitrogen treatment, the frozen tumor-bearing nerve graft was trimmed to a 5-mm-long tissue and implanted into another F344/NJcl-rnu/rnu rat, in which a 5-mm-long section of the sciatic nerve was resected to create a nerve gap. Experiment 2 was performed with 12 rats; six rats were evaluated at 24 and 48 weeks postoperatively. To assess nerve regeneration and tumor recurrence after nerve reconstruction using frozen tumor-bearing nerve grafts obtained from the nude rat with human fibrosarcoma involving the sciatic nerve, the sciatic nerve's function and histologic findings were evaluated in the same way as in Experiment 1.

Results: Experiment 1: The lower limb muscle volume decreased once at 4 weeks in the autograft and frozen autograft groups and gradually increased thereafter. The tibialis anterior muscle wet weight ratio, sciatic functional index, muscle fiber size, and electrophysiologic evaluation showed higher nerve regeneration potential in the autograft and frozen autograft groups than in the resection group. The median S100-positive areas (interquartile range [IQR]) in the autograft group were larger than those in the frozen autograft group at 12 weeks (0.83 [IQR 0.78 to 0.88] versus 0.57 [IQR 0.53 to 0.61], difference of medians 0.26; p = 0.04) and at 48 weeks (0.86 [IQR 0.83 to 0.99] versus 0.74 [IQR 0.69 to 0.81], difference of median 0.12; p = 0.03). Experiment 2: Lower limb muscle volume decreased at 4 weeks and gradually increased thereafter. The median muscle fiber size increased from 0.89 (IQR 0.75 to 0.90) at 24 weeks to 1.20 (IQR 1.08 to 1.34) at 48 weeks (difference of median 0.31; p< 0.01). The median amplitude increased from 0.60 (IQR 0.56 to 0.67) at 24 weeks to 0.81 (IQR 0.76 to 0.90) at 48 weeks (difference of median 0.21; p < 0.01). Despite tumor involvement and freezing treatment, tumor-bearing frozen grafts demonstrated nerve regeneration activity, with no local recurrence observed at 48 weeks postoperatively in nude rats.

Conclusion: Tumor-bearing frozen nerve grafts demonstrated nerve regeneration activity, and there was no tumor recurrence in rats in vivo.

Clinical relevance: A frozen nerve autograft has a similar regenerative potential to that of a nerve autograft. Although the findings in a rat model do not guarantee efficacy in humans, if they are substantiated by large-animal models, clinical trials will be needed to evaluate the efficacy of tumor-bearing frozen nerve grafts in humans.

Fig. 1

This flowchart shows an overview of this experiment. This experiment was performed in two parts: an experiment to determine the effect of liquid nitrogen treatment on limb function (Experiment 1) and an experiment to investigate tumor recurrence and function after reconstruction using a tumor-bearing frozen graft (Experiment 2). In Experiment 1, we used Wistar rats to generate three models: an autograft transplantation model (autograft group), a frozen autograft transplantation model (frozen autograft group), and a no-reconstruction model (resection group). Twenty-four rats were evaluated in each group, and six rats per group were evaluated at 4, 12, 24, and 48 weeks postoperatively. In Experiment 2, we used nude rats (F344/NJCrl-rnu/rnu) and obtained tumor-bearing nerve grafts. Twelve rats were included in Experiment 2, and six rats were evaluated at 24 and 48 weeks postoperatively. SFI = sciatic functional index.

Fig. 2

This figure shows the procedure in Experiment 1. (A) The sciatic nerve of a Wistar rat before resection is shown. (B) A 10-mm-long nerve autograft was obtained. (C) An autograft transplantation model was developed in which the resected sciatic nerve was reversed and sutured in place. (D) The nerve gap was not reconstructed in the resection group.

Fig. 3

This figure shows the procedure in Experiment 2. (A) HT1080 cells were injected near the bilateral sciatic nerves of a nude rat through a small incision. (B) The injected rat exhibited bilateral sciatic nerve palsy after 2 weeks. (C-D) The sciatic nerve with a tumor was resected. (E) The sciatic nerve (arrow) was surrounded by a tumor. (F) Hematoxylin and eosin staining confirmed that the nerve was surrounded by the tumor (bar = 300 µm). (G) The tumor-bearing nerve was treated with liquid nitrogen. (H) The tumor-bearing frozen graft was trimmed to 5 mm and implanted into another nude rat.

Fig. 4

Axial CT data were analyzed using a 3D image analyzer (Synapse Vincent, Fujifilm Inc).

Fig. 5

The results of Experiment 1 are shown. (A) The lower limb muscle volume of the autograft and frozen autograft groups was higher than that of the resection group after 8 weeks. (B) The autograft group had a higher SFI than the resection group after 24 weeks. (C) The latency in the autograft and frozen autograft groups improved after 12 weeks. (D) The amplitude in the autograft group was higher than that in the resection group. The frozen autograft group had higher amplitude than the resection group after 12 and 48 weeks.

Fig. 6

(A) The anterior tibialis muscles of rats were stained with Masson trichrome stain in Experiment 1 (bar = 100 µm). (B) The muscle fiber size was calculated with Masson trichrome staining. The autograft and frozen autograft groups had larger muscle fibers than the resection group after 12 weeks. (C) The muscle wet weight ratio is shown. The autograft and frozen autograft groups had a higher muscle wet weight ratio than the resection group after 12 weeks.

Fig. 7

Immunostaining was performed in implanted nerves in Experiment 1 (bar = 100 µm). (A) S100 protein staining is shown. The control group included an intact sciatic nerve (normal side) that was diffusely positive for the S100 protein. In the autograft and frozen autograft groups, the S100-positive area gradually increased. (B) Neurofilament staining is shown. The control group had an intact sciatic nerve (normal side) and displayed positive neurofilament staining in the central nerve. In the autograft and frozen autograft groups, the neurofilament-positive area gradually increased.

Fig. 8

This figure shown the S100 area and neurofilament-positive areas in Experiment 1. (A) The S100-positive area in the autograft group was higher at 12 and 48 weeks than that in the frozen autograft group. (B) There was no difference in the neurofilament-positive area in the autograft and frozen autograft groups.

Fig. 9

The results of Experiment 2 are shown. (A) Lower limb muscle volume was calculated using CT data. The lower limb’s muscle volume initially decreased at 4 weeks and then gradually increased. (B) The SFI and muscle fiber size, calculated by (C) Masson trichrome staining, were higher at 48 weeks than at 24 weeks. We performed an electrophysiologic evaluation of (D) latency and (E) amplitude. An immunohistochemical evaluation of the (F) S100-positive area and (G) neurofilament-positive area was performed. The S100-positive area was higher at 48 weeks than at 24 weeks.