Objective To investigate the feasibility of establishing the visualization models of intraneural microvessels of sciatic nerves in Sprague Dawley (SD) rats by systemic infusion of Evan’s blue (EB) or lead oxide and to compare the advantages and disadvantages. Methods Fifteen healthy adult SD rats of either gender, weighing 200-250 g, were randomly divided into traditional group (group A, n=5), fluorescence group (group B, n=5), and radiography group (group C, n=5). Ink, EB, and lead oxide, all mixed with gelatin solution, were injected in groups A, B, and C, respectively. After 2 hours of cryopreservation under 4°C, all sciatic nerves were harvested and observed through stereomicroscope to make sure the filling condition. The two-dimentional (2D) images were then collected via reflexion fluorescent microscope in group B and via micro-CT scan in group C. All images were imported into computer to establish three-dimentional (3D) reconstruction models by Mimics 15.0. Results All groups could show the outline of intraneural microvessels of sciatic nerves under stereomicroscope. Diameters of them were measured under fluorescent microscope, ranging from 10 µm to 30 µm. Both groups B and C could establish 3D reconstruction models from 2D images. These models could clearly reproduce the structure of microvessels. Conclusion Both EB and lead oxide can be used to establish 3D reconstruction models to observe structure of the intraneural vessels. However, EB has some disadvantages, such as predisposition to infiltration, grainy 2D images and time-consuming procedure; it is not suitable for researches of large specimen. Though 2D pictures from lead oxide have lower resolution than EB, it is easier to be manipulated and appropriate for experiments of large specimen.
Objective To observe the systemic and local immune response after repair of nerve defect with acellular nerve xenograft laden with allogenic adipose-derived stem cells (ADSCs) in rhesus monkey so as to evaluate the safety of the proposed material for nerve reconstruction. Methods Bilateral tibial nerves were taken from a healthy adult male landrace (weighing 48 kg) to prepare acellular nerve xenograft by chemical extraction. ADSCs were isolated from a healthy adult male rhesus monkey (weighing 4.5 kg), and were seeded into the acellular nerve grafts. The radial nerve defect models with 25 mm in length were established in 10 healthy adult female rhesus monkeys (weighing 3-5 kg), and they were divided into cell-laden group (n=5) and non-cell-laden group (n=5) randomly. Defect was repaired with acellular nerve xenograft laden with allogenic ADSCs in cell-laden group, with acellular nerve xenograft only in non-cell-laden group. The blood samples were taken from peripheral vein preoperatively and at 14, 60, and 90 days after operation for lymphocyte analysis; at 5 months after operation, the grafts were harvested to perform histological examination for local immune response and nerve regeneration. The nerve autograft in rhesus monkey was used as control. Results In cell-laden group and non-cell-laden group, no significant difference was found in the count of lymphocytes and T lymphocytes, the percentage of T lymphocytes, CD8+ T lymphocytes, as well as the ratio of CD4+ T lymphocytes to CD8+ T lymphocytes between pre- and post-operation (P gt; 0.05); in cell-laden group, the percentage of CD4+ T lymphocytes at 14 days was significantly lower than that at 60 and 90 days postoperatively (P lt; 0.05). The percentage of CD4+ T lymphocytes in cell-laden group was significantly lower than that in non-cell-laden group at 14 days (P lt; 0.05), but no significant difference was found in the other indexes at the other time between 2 groups (P gt; 0.05). At 5 months after operation, mild adhesion was found on the surface of nerve xenografts; the epineurium of nerve xenografts was thicker than that of nerve autografts; and neither necrosis nor fibrosis was found. CD3+, CD4+, CD8+, CD68+, and CD163+ T lymphocytes were scattered within the grafts, in which regenerative axons were revealed. CD3+, CD4+, CD8+, CD68+, and CD163+ T lymphocytes were comparable in cell-laden group, non-cell-laden group, and autograft group. Conclusion Repair of nerve defect with acellular nerve xenograft elicits neither systemic nor local immune response in rhesus monkeys. Implantation of allogenic ADSCs might result in transient depression of CD4+ T lymphocytes proliferation early after surgery, no immune response can be found.