An experimental model of proliferative vitretinopathy(PVR) induced by macrophages was used for the evaluation of drug efficacy of daunomycin encapsulated in liposomes in the treatment of PVR.Five mu;g daunomycin(n=40),10mu;g daunomycin-liposome(DL,n=30)and 0.1 ml saline or empty liposomes(n=40,as controls)were injected into the rabbit vitreous after macrophage injection.Retinal detachment developed in 77.5% of the control eyes on day 28,compared to 33.3% of the eyes treated with DL(P＜0.01)and 50% of the daunomycin-treated eyes(P＜0.05).The results suggest that encapsulation in liposomes of cytotoxic agents can enhance drug efficacy.The phasic course of development of PVR is important in the selection of particular drugs. （Chin J Ocul Fundus Dis,1993,9:77-80）
Objective To realize the visualization of three-dimensional microstructure of rabbit sciatic nerve bundles by micro-CT and three-dimensional visualization software Mimics17.0. Methods The sciatic nerve tissues from 6 New Zealand rabbits were divided into 2 groups (n=3), and the sciatic nerve tissues were stained by 1% (group A) and 5% (group B) Lugol solution respectively. After staining for 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 hours, the imaging changes of specimens were observed by light microscope and micro-CT. The clear micro-CT images were exported to the Mimics software to complete the visualization of three-dimensional microstructure of rabbit sciatic nerve according to three-dimensional reconstruction tool. Results The clear three-dimensional microstructure images could be observed in group A at 2.5 hours after staining and in group B at 1.5 hours after staining by light microscope and micro-CT. The sciatic nerve of New Zealand rabbits were divides into 3 bundles and each of them was relatively fixed. There was no obvious crossing or mergers between each bundle. The cross-sectional area of each bundle was (0.425±0.013), (0.038±0.007), and (0.242±0.026) mm2 respectively. The digital model could clearly reflect the microstructure of the sciatic nerve at all cross sections. Conclusion The internal structure of New Zealand rabbits sciatic nerve can be clearly reflected by micro-CT scanning. It provides a reliable method for establishing a nerve microstructure database with large amount specimens.
ObjectiveTo investigate the early effects of acellular xenogeneic nerve combined with adipose-derived stem cells (ADSCs) and platelet rich plasma (PRP) in repairing facial nerve injury in rabbits.MethodsThe bilateral sciatic nerves of 15 3-month-old male Sprague-Dawley rats were harvested and decellularized as xenografts. The allogeneic ADSCs were extracted from the neck and back fat pad of healthy adult New Zealand rabbits with a method of digestion by collagenase type Ⅰ and the autologous PRP was prepared by two step centrifugation. The 3rd generation ADSCs with good growth were labelled with CM-Dil living cell stain, and the labelling and fluorescence attenuation of the cells were observed by fluorescence microscope. Another 32 New Zealand rabbits were randomly divided into 4 groups and established the left facial nerve defect in length of 1 cm (n=8). The nerve defects of groups A, B, C, and D were repaired with CM-Dil-ADSCs composite xenogeneic nerve+autologous PRP, CM-Dil-ADSCs composite xenogeneic nerve, xenogeneic nerve, and autologous nerve, respectively. At 1 and 8 weeks after operation, the angle between the upper lip and the median line of the face (angle θ) was measured. At 4 and 8 weeks after operation, the nerve conduction velocity was recorded by electrophysiological examination. At 8 weeks after operation, the CM-Dil-ADSCs at the distal and proximal ends of regenerative nerve graft segment in groups A and B were observed by fluorescence microscopy; after toluidine blue staining, the number of myelinated nerve fibers in regenerated nerve was calculated; the structure of regenerated nerve fibers was observed by transmission electron microscope.ResultsADSCs labelled by CM-Dil showed that the labelling rate of cells was more than 90% under fluorescence microscope, and the labelled cells proliferated well, and the fluorescence attenuated slightly after passage. All the animals survived after operation, the incision healed well and no infection occurred. At 1 week after operation, all the animals in each group had different degrees of dysfunction. The angle θ of the left side in groups A, B, C, and D were (53.4±2.5), (54.0±2.6), (53.7±2.4), and (53.0±2.1)°, respectively; showing significant differences when compared with the healthy sides (P<0.05). At 8 weeks after operation, the angle θ of the left side in groups A, B, C, and D were (61.9±4.7), (56.8±4.2), (54.6±3.8), and (63.8±5.8)°, respectively; showing significant differences when compared with the healthy sides and with the values at 1 week (P<0.05). Gross observation showed that the integrity and continuity of regenerated nerve in 4 groups were good, and no neuroma and obvious enlargement was found. At 4 and 8 weeks after operation, the electrophysiological examination results showed that the nerve conduction velocity was significantly faster in groups A and D than in groups B and C (P<0.05), and in group B than in group C (P<0.05); no significant difference was found between groups A and D (P>0.05). At 8 weeks after operation, the fluorescence microscopy observation showed a large number of CM-Dil-ADSCs passing through the distal and proximal transplants in group A, and relatively few cells passing in group B. Toluidine blue staining showed that the density of myelinated nerve fibers in groups A and D were significantly higher than those in groups B and C (P<0.05), and in group B than in group C (P<0.05); no significant difference was found between groups A and D (P>0.05). Transmission electron microscope observation showed that the myelinated nerve sheath in group D was large in diameter and thickness in wall. The morphology of myelin sheath in group A was irregular and smaller than that in group D, and there was no significant difference between groups B and C.ConclusionADSCs can survive as a seed cell in vivo, and can be differentiated into Schwann-like cells under PRP induction. It can achieve better results when combined with acellular xenogeneic nerve to repair peripheral nerve injury in rabbits.
Objective To study the effect of two cytokines, basic fibroblast growth factor(bFGF) and insulin-like growth factor-I(IGF-I), on cell proliferation in chondrocytes of adult rabbits. Methods The primary chondrocytes of adult rabbits were harvested and cultured with bFGF and IGF-I at different concentrations,respectively, as well as with the mixture of the two cytokines; the quantity of cultured chondrocytes was detected by MTT assay at the 24th, 48th and 72th hours; and the final fold increase of different groups was measured by cell count for the 3rd passage; and the proliferation index of the groups was recorded by flowing cytometer on the 14th day. Results ① The cultured chondrocytes with either bFGF, IGF-I or their mixture were significantly more than that of control group at the 24th, 48th and 72th hours (P＜0.01). ② After the 3rd passage, the final folds of proliferation were significantly higher in the groups with cytokinesthan in the control group (P＜0.01); and the final fold with the mixture ofcytokines was significantly higher than that of both IGF-I and bFGF (P＜0.01). ③ Theproliferation index was significantly higher in the groups with cytokines than in the control group (P＜0.01); the proliferation index with the mixture of cytokines was significantly higher than that of both IGF-I and bFGF (P＜0.05); besides, proliferation index was higher when cytokine was applied twice than once (P＜0.05). Conclusion bFGF and IGF-I could promote chondrocytes proliferation of adult rabbits obviously and they are synergistic in cell proliferation.
ObjectiveTo study the effect of intercellular adhesion (ica) operon of Staphylococcus epidermidis on the inflammation associated with mixed biofilm of Staphylococcus epidermidis and Candida albicans on endotracheal tube material in rabbits. MethodsThe standard strains of Staphylococcus epidermidis RP62A (ica operon positive, positive group) and ATCC12228 (ica operon negative, negative group) were taken to prepare a bacterial solution with a concentration of 1×106 CFU/mL, respectively. Then, the two bacterial solutions were mixed with the standard strain of Candida albicans ATCC10231 of the same concentration to prepare a mixed culture solution at a ratio of 1∶1, respectively. The mixed culture solution was incubated with endotracheal tube material for 24 hours. The formation of mixed biofilm on the surface of the material was observed by scanning electron microscope. Thirty New Zealand rabbits, aged 4-6 months, were divided into two groups (n=15), and the endotracheal tube materials of the positive group and the negative group that were incubated for 24 hours were implanted beside the trachea. The body mass of rabbits in the two groups was measured before operation and at 1, 3, and 7 days after operation. At 1, 3, and 7 days after operation, the levels of interleukin 1β (IL-1β), IL-6, tumor necrosis factor α (TNF-α), and monocytechemotactic protein 1 (MCP-1) were detected by using an ELISA test kit. At 7 days after operation, the formation of mixed biofilm on the surface of the endotracheal tube materials was observed by scanning electron microscope, the inflammation and infiltration of tissues around the materials were observed by HE staining, and the bacterial infections in heart, lung, liver, and kidney were observed by plate colony counting method.ResultsScanning electron microscope observation showed that the mixed biofilm structure was obvious in the positive group after 24 hours in vitro incubation, but no mixed biofilm formation was observed in the negative group. In vivo studies showed that there was no significant difference in body mass between the two groups before operation and at 1, 3, and 7 days after operation (P>0.05). Compared with the negative group, the levels of MCP-1 and IL-1β at 1 day, and the levels of IL-1β, MCP-1, IL-6, and TNF-α at 3 and 7 days in the positive group all increased, with significant differences (P<0.05). Scanning electron microscope observation showed that a large amount of Staphylococcus epidermis and mixed biofilm structure were observed in the positive group, and a very small amount of bacteria was observed in the negative group with no mixed biofilm structure. HE staining of surrounding tissue showed inflammatory cell infiltration in both groups, and neutrophils and lymphocytes were more in the positive group than in the negative group. There was no significant difference in the number of bacterial infections in heart and liver between the two groups (P>0.05). The number of bacterial infections in lung and kidney in the positive group was higher than that in negative group (P<0.05).ConclusionIn the mixed infection of Staphylococcus epidermidis and Candida albicans, the ica operon may strengthen the structure of the biofilm and the spread of the biofilm in vivo, leading to increased inflammatory factors, and the bacteria are difficult to remove and persist.
ObjectiveTo investigate the mechanism of early vascularization of the tissue engineered bone in the treatment of rabbit radial bone defect by local injection of angiopoietin 2 (Ang-2).MethodsForty-eight New Zealand white rabbits were established unilateral 1.5 cm long radius defect models. After implantation of hydroxyapatite/collagen scaffolds in bone defects, the rabbits were randomly divided into 2 groups: control group (group A) and Ang-2 group (group B) were daily injected with 1 mL normal saline and 1 mL saline-soluble 400 ng/mL Ang-2 at the bone defect within 2 weeks after operation, respectively. Western blot was used to detect the expressions of autophagy related protein [microtubule associated protein 1 light chain 3 (LC3), Beclin-1], angiogenesis related protein [vascular endothelial growth factor (VEGF)], and autophagy degradable substrate protein (SQSTMl/p62) in callus. X-ray films examination and Lane-Sandhu X-ray scoring were performed to evaluate the bone defect repair at 4, 8, and 12 weeks after operation. The rabbits were sacrificed at 12 weeks after operation for gross observation, and the angiogenesis of bone defect area was observed by HE staining.ResultsWestern blot assay showed that the relative expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1, and VEGF in group B were significantly higher than those in group A, and the relative expression of SQSTMl/p62 was significantly lower than that in group A (P<0.05). Radiographic and gross observation of specimens showed that only a few callus were formed in group A, the bone defect was not repaired; more callus were formed and complete repair of bone defect was observed in group B. The Lane-Sandhu scores in group B were significantly higher than those in group A at 4, 8, and 12 weeks after operation (P<0.05). HE staining showed that the Harvard tubes in group B were well arranged and the number of new vessels was significantly higher than that in group A (t=–11.879, P=0.000).ConclusionLocal injection of appropriate concentration of Ang-2 may promote early vascularization and bone defect repair of tissue engineered bone in rabbits by enhancing autophagy.
Objective To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups (n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes (P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers (P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B (P<0.05), and the level of TIMP-1 in group C was significantly higher (P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B (P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B (P<0.05).Conclusion CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.
Objective To investigate the effect of Kartogenin (KGN) combined with adipose-derived stem cells (ADSCs) on tendon-bone healing after anterior cruciate ligament (ACL) reconstruction in rabbits. Methods After the primary ADSCs were cultured by passaging, the 3rd generation cells were cultured with 10 μmol/L KGN solution for 72 hours. The supernatant of KGN-ADSCs was harvested and mixed with fibrin glue at a ratio of 1∶1; the 3rd generation ADSCs were mixed with fibrin glue as a control. Eighty adult New Zealand white rabbits were taken and randomly divided into 4 groups: saline group (group A), ADSCs group (group B), KGN-ADSCs group (group C), and sham-operated group (group D). After the ACL reconstruction model was prepared in groups A-C, the saline, the mixture of ADSCs and fibrin glue, and the mixture of supernatant of KGN-ADSCs and fibrin glue were injected into the tendon-bone interface and tendon gap, respectively. ACL was only exposed without other treatment in group D. The general conditions of the animals were observed after operation. At 6 and 12 weeks, the tendon-bone interface tissues and ACL specimens were taken and the tendon-bone healing was observed by HE staining, c-Jun N-terminal kinase (JNK) immunohistochemical staining, and TUNEL apoptosis assay. The fibroblasts were counted, and the positive expression rate of JNK protein and apoptosis index (AI) were measured. At the same time point, the tensile strength test was performed to measure the maximum load and the maximum tensile distance to observe the biomechanical properties. Results Twenty-eight rabbits were excluded from the study due to incision infection or death, and finally 12, 12, 12, and 16 rabbits in groups A-D were included in the study, respectively. After operation, the tendon-bone interface of groups A and B healed poorly, while group C healed well. At 6 and 12 weeks, the number of fibroblasts and positive expression rate of JNK protein in group C were significantly higher than those of groups A, B, and D (P<0.05). Compared with 6 weeks, the number of fibroblasts gradually decreased and the positive expression rate of JNK protein and AI decreased in group C at 12 weeks after operation, with significant differences (P<0.05). Biomechanical tests showed that the maximum loads at 6 and 12 weeks after operation in group C were higher than in groups A and B, but lower than those in group D, while the maximum tensile distance results were opposite, but the differences between groups were significant (P<0.05). Conclusion After ACL reconstruction, local injection of a mixture of KGN-ADSCs and fibrin glue can promote the tendon-bone healing and enhance the mechanical strength and tensile resistance of the tendon-bone interface.
Objective To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) (NNSCS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes in vitro. Methods Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with NNSCS to form NNSCS/pDNA complexes, respectively. Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits. The second generation chondrocytes were divided into 3 intervention groups: normal cell control group (group A), NNSCS/GV268 empty plasmid transfection group (group B), and NNSCS/GV268-miR-140 transfection group (group C). NNSCS/GV268 and NNSCS/GV268-miR- 140 complexes were transiently transfected into cells of groups B and C. After transfection, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140; Annexin Ⅴ-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes; the expressions of Sox9, Aggrecan, and histone deacetylase 4 (Hdac4) were detected by RT-qPCR. Results RT-qPCR showed that the expression of miR-140 in group C was significantly higher than that in groups A and B (P<0.05). Compared with groups A and B, the apoptosis rate in group C was decreased and the proliferation activity was improved, Sox9 and Aggrecan gene expressions were significantly up-regulated, and Hdac4 gene expression was significantly down-regulated (P<0.05). There was no significant difference in above indexes between groups A and B (P>0.05). Conclusion Exogenous gene can be carried into the chondrocytes by NNSCS and expressed efficiently, the high expression of miR-140 can improve the biological activity of chondrocytes cultured in vitro, which provides important experimental basis for the treatment of cartilage damage diseases.
Objective To investigate the influence of colectomy on the expressions of 5-hydroxy tryptamine (5-HT) and chromogranin A (CgA) in colon mucosa of Chinchilla rabbits. Methods Colon (7-8 cm) upon colon-rectum junction (control group) of 15 Chinchilla rabbits was cut out. After two weeks, these rabbits were executed and the samples of colon at anastomotic stoma (study group) were taken. 5-HT positive cells and CgA positive cells in two groups were detected by immunohistochemical method. Results The number of 5-HT positive cells was 10.40±2.22 in control group， and 26.27±2.35 in study group; the number of CgA positive cells was 20.60±5.34 in control group, and 51.51±6.13 in study group. There were significant differences between two groups respectively (P＜0.01). Conclusion The increase of 5-HT positive cells and CgA positive cells can be caused by colectomy.