This study aims at the evaluation of blood vessel reconstruction process of decellularized small diameter vessels prepared by a hyperosmotic electrolyte solution treatment not only histologically but also physiologically in rat transplantation model. Complete cell removal by a hyperosmotic electrolyte solution treatment was confirmed by hematoxylin/eosin staining and scanning electron microscopic observation. All acellular vessels transplanted into the rat abdominal aorta were patent up to 14 months. One week post-transplantation, the vWF-positive cells were observed on the luminal surface but the layer formation did not complete. Five weeks following transplantation, the vWF-positive endothelial cells were located on the intima consistent with intact endothelial cells. Beneath the endothelial cells, α-SMA-positive smooth muscle cells were distributed. The harvested vessels displayed formation of tunica intima (endothelial cells) and tunica medulla (smooth muscle cell) layers. We also examined the physiological properties of the vessels 12 months post-transplantation using a wire myograph system. The transplanted vessels contracted upon addition of norepinephrine and relaxed upon addition of sodium nitroprusside as well as the native vessels. In conclusion, the acellular vessels prepared with hyperosmotic electrolytic solution showed excellent and long-term patency, which may be related to the successful preservation of vascular ECM. In addition, the acellular vessels revealed the intima/medulla regeneration with the physiological contraction-relaxation functions in response to the each substance.
Our methodology preserves the ECM, is simple to develop, and does not involve substances that harm biogenic tissue. Acellular nerve tissue processed in this way could become a substitute material for bridging nerve gaps. Our method could also aid in the development of other acellular tissues.
a b s t r a c tIntroduction: Several methods of nerve reconstruction for facial nerve palsy are known. Although the recently introduced method of "cross-linking" of the facial and hypoglossal nerves with a grafted nerve has proved efficacious, the underlying mechanism is unclear. Methods: In this study, we created an animal model with Wistar rats and analyzed the newly reconstructed neural circuit by anterograde and retrograde neural tracer methods. The saphenous nerve was harvested as a graft, and its double end-to-side neurorrhaphy with the facial and hypoglossal nerves with epineural windows was carried out under the microscope. After an appropriate interval, small amounts of fluoro-ruby or fluoro-emerald were injected into the animals and analyzed 5 days later by fluorescent microscopy (Anterograde experiment: fluoro-ruby into the hypoglossal nucleus at 5 weeks; retrograde experiment: fluoro-ruby into the distal facial nerve sheath and fluoro-emerald into the distal hypoglossal nerve sheath, both at two months.). Results: The labeled axons derived from the hypoglossal nucleus were observed passing through the grafted nerve to the facial nerve. On the other hand, retrogradely labeled neurons were observed at both the hypoglossal and facial nuclei with some double-labeled neurons, suggesting that collateral sprouting had occurred. Conclusions: We suggest that the newly constructed neural circuits we observed are conducive to the treatment of facial nerve palsy.
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