The pathogensis of choroidal vascular changes in traumatic rtinopethy remains uncertain.We performed scanning electron micrmcopie (SEM) observation of methyl methalerylare vascular corrosion casts in a rabbi model with severe retinal contusion. Areas of filling defects in corrosion casts of the choriocapillaries, correspending to the areas of impact retinal lesions were noted in the traumatized eyes one to 28 days after trauma.No neovascularization was found in the eyes 56 days after trauma. The results confirm that obstruction and disappearance of involved choriocapillaries are the main changes of choroidal vasculatrue in severe blunt tram. The changes may be associated with continuous necrosis, of the photoreceptors 4 weeks after injury. （Chin J Ocul Fundus Dis,1993,9:5-7）
Six New Zealand white rabbits were under the pentobarbital anesthesia, the sciatic nerve trunk were exposed and divided. Afterwards, both the proximal and distal ends of the divided nerve trunk were incubated with Blue-SAb in the micropipe for 30 minutes at room temperature. The sensory fascieuli which were bly Blue-immunostained among unstained fasciculi and other tissues had been seen under the magnifying operating microscope (×4). The neural cells of the spinal cord and the ganglion were cultured on RPMI 1640 medium containing Bright- Blue.The growth and the metabolism of the neural cells were tested by MTT method. The t-test was used to determine the statistical difference blue staining of the neural cells was considered to be of no statistical significance (Pgt;0.05).
Objective To study the mechanism of restenosis of the vein graft and the effect of the grafting injury to the vein graft. Methods One side of the 36 healthy rabbits was randomly chosen as the V-A group, and on the side a 1.5cmlong femoral vein was obtained, and an 0.5-cm-long segment of the obtained femoral vein was separated as the control group. The remaining 1-cm-long femoral vein was inverted and was autogenously implanted into the femoral artery on the same side of the rabbit. The other side of the rabbits was chosen as the V-V group, and on this side a 1-cm-long femoral vein was obtained ex vivo and then was sutured in situ. The vein grafts on both sides were harvested 4 weeks after operation. The specimens from the harvested vein grafts were stained with HE and theelastic fiber Victoria blue for an observation on the histological changes in the walls of the vein grafts, and the specimens were also stained by the immunohistochemistry of the proliferating cell nuclear antigen (PCNA) for an observation on the wall cell proliferation of the vein grafts. The changes in the ultrastructure of the proliferated wall cells of the vein grafts were observed under electron microscope. The two sides of the rabbits were compared. Results The smooth muscle cells of the media developed hyperplasia, but theintima and the media remained unchanged in their thickness (3.50±0.41 μm, 12.23±1.59 μm) in the V-V group, with no difference when compared with the control group (3.40±0.37 μm, 12.14±1.62 μm); however, when compared with the V-A group (25.60±3.21 μm, 21.30±2.47 μm),there was a significant difference in the thickness (Plt;0.01). There were no cells positive for PCNA by the immunohistochemistry examination in the control group. The cells positive for PCNA were found in the intima and the media in both the V-V group and the V-A group; however, the percentageof the cells positive for PCNA in the intima and the media was significantly greater in the V-A group than in the V-V group (16.4%±1.9% and 36.5%±3.7% vs 5.9%±1.3% and 23.4%±3.4%, Plt;0.01). In the V-V group, the endothelial cell could be observed under transmis-sion electron microscope, which was flat and had a processlike villus at its free end, and the endothelial cells were closely arranged andhad hyperplasia of the smooth muscle cells in the media. But in the V-A group,the endothelial cells had an obvious hyperplasia with an irregular shape and a widened space between the cells, and in the intima a great amount of the smooth muscle cells could be observed, which had a broken basement membrane. The smooth muscle cells also had an obvious hyperplasia in the media. The shape and alignment of the endothelial cells in the control group were similar to those in the V-V group, but the hyperplasia of the smooth muscle cells was not observed in the media. Conclusion The grafting injury can cause hyperplasia ofthe vascular wall cells, and if the hemodynamics is changed simultaneously, more serious hyperplasia and cell migration can be observed from the media to the intima, resultingin restenosis of the blood vessels. So, if we can reduce the grafting injury and improve the microcirculation of the vein graft, we may find out the methods ofpreventing restenosis of the vein graft. The animal model of the V-V graftcan help to understand the mechanism of restenosis of the vein graft.
One eye each in 3 groups of 12 pigmented rabbits after bilateral vitrectomy received 0.5mg, 1mg or 2mg triamcinolone acetonide (TA), respectively. The fellow eye received only balance saline solution as control. Ophthalmoscopy and electroretinography were performed during 1 day to 38 days after vitrectomy and drug injection. Light and electronmicroscopic studies were done on the 28th day. The particles of drug were visible on day 28 in all TA-treated eyes. Administration of 0. 5rug and 1mg TA did not result in different changes in ERG b-wave amplitudes compared with those in control eyes(P＞0. 05). There were significant elevations of ERG b-wave in 2mg TA eyes compared to the control eyes(Plt;0.05), Both ligbt and electronmicroscopy of the retina in these groups were almost normal. The results showed no Toxielties in TA treated eye up to 2mg after vitrectomy. This offers the experimental evidence as a baseline for combining TA with vitrectomy to reduce recurrence of proliferative vitreoretinopathy. （Chin J Ocul Fundus Dis,1996,12: 105- 107）
Objective To evaluate the feasibility and validity of chondrogenic differentiation of marrow clot after microfracture of bone marrow stimulation combined with bone marrow mesenchymal stem cells (BMSCs)-derived extracellular matrix (ECM) scaffold in vitro. Methods BMSCs were obtained and isolated from 20 New Zealand white rabbits (5-6 months old). The 3rd passage cells were cultured and induced to osteoblasts, chondrocytes, and adipocytes in vitro, respectively. ECM scaffold was manufactured using the 3rd passage cells via a freeze-dying method. Microstructure was observed by scanning electron microscope (SEM). A full-thickness cartilage defect (6 mm in diameter) was established and 5 microholes (1 mm in diameter and 3 mm in depth) were created with a syringe needle in the trochlear groove of the femur of rabbits to get the marrow clots. Another 20 rabbits which were not punctured were randomly divided into groups A (n=10) and B (n=10): culture of the marrow clot alone (group A) and culture of the marrow clot with transforming growth factor β3 (TGF-β3) (group B). Twenty rabbits which were punctured were randomly divided into groups C (n=10) and D (n=10): culture of the ECM scaffold and marrow clot composite (group C) and culture of the ECM scaffold and marrow clot composite with TGF-β3 (group D). The cultured tissues were observed and evaluated by gross morphology, histology, immunohistochemistry, and biochemical composition at 1, 2, 4, and 8 weeks after culture. Results Cells were successfully induced into osteoblasts, chondrocytes, and adipocytes in vitro. Highly porous microstructure of the ECM scaffold was observed by SEM. The cultured tissue gradually reduced in size with time and disappeared at 8 weeks in group A. Soft and loose structure developed in group C during culturing. Chondroid tissue with smooth surface developed in groups B and D with time. The cultured tissue size of groups C and D were significantly larger than that of group B at 4 and 8 weeks (P lt; 0.05); group D was significantly larger than group C in size (P lt; 0.05). Few cells were seen, and no glycosaminoglycan (GAG) and collagen type II accumulated in groups A and C; many cartilage lacunas containing cells were observed and more GAG and collagen type II were synthesized in groups B and D. The contents of GAG and collagen increased gradually with time in groups B and D, especially in group D, and significant difference was found between groups B and D at 4 and 8 weeks (P lt; 0.05). Conclusion The BMSCs-derived ECM scaffold combined with the marrow clot after microfracture of bone marrow stimulation is effective in TGF-β3-induced chondrogenic differentiation in vitro.
Objective To evaluate the cell biological features and the effect of transplantation of transforming growth factor β3 (TGF-β3) gene-modified nucleus pulposus (NP) cells on the degeneration of lumbar intervertebral discs in vitro. Methods NP cells at passage 2 were infected by recombinant adenovirus carrying TGF-β3 (Ad-TGF-β3) gene (Ad-TGF-β3 group), and then the cell biological features were observed by cell vital ity assay, the expression of the TGF-β3 protein was determined by Western blot, the expression of collagen type II in logarithmic growth phase was determined by immunocytochemistry. The cells with adenovirus-transfected (Adv group) and the un-transfected cells (blank group) were used as controls. The model of lumbar disc degeneration was establ ished by needl ing L3, 4, L4, 5, and L5, 6 in 30 New Zealand rabbits (weighing 3.2-3.5 kg, male or female). Then Ad-TGF-β3-transfected rabbit degenerative nucleus pulposus cells (100 μL, 1 × 105/ mL, group A, n=12), no gene-modified nucleus pulposus cells (100 μL, 1 × 105/mL, group B, n=12), and phosphatebuffered sal ine (PBS, 100 μL, group C, n=6) were injected into degenerative lumbar intervertebral discs, respectively. L3, 4, L4, 5, and L5, 6 disc were harvested from the rabbits (4 in groups A and B, 2 in group C) at 6, 10, and 14 weeks respectively to perform histological observation and detect the expression of collagen type II and proteoglycan by RT-PCR. Results The viabil ity of nucleus pulposus cells was obviously improved after transfected by recombinant Ad-TGF-β3 gene. At 3, 7, and 14 days after transfected, TGF-β3 expression gradually increased in nucleus pulposus cells. The positive staining of collagen type II was seen in Ad-TGF-β3 group, and the positive rate was significantly higher than that of Adv group and blank group (P lt; 0.05). The disc degeneration in group A was sl ighter than that in groups B and C. The expressions of collagen type II mRNA and proteoglycan mRNA in group A were significantly higher than those in groups B and C at 6, 10, and 14 weeks (P lt; 0.05). Conclusion TGF-β3 can improve the biological activity of NP cells and promote the biosynthesis of collagen type II and proteoglycan in intervertebral discs, alleviate the degeneration of intervertebral discs after transplantation.
Objective To examine the effects of alendronate (ALN) on IL-1β-stimulated chondrocyte of rabbit in vitro and on cartilage and subchondral bone in rabbit osteoarthritis (OA) induced by anterior cruciate l igament transection (ACLT). Methods The chondrocytes from articular surface of healthy 3-month-old Japanese White rabbits were obtained by the method of enzyme digestion and cultured in vitro. The third generation chondrocytes were assigned into three groups: thechondrocytes were cultured in DMEM medium with 10 ng/mL IL-1β for 2 days, subsequently with (ALN group, group A1) orwithout (IL-1β group, group B1) 1 × 10-6 mol/L ALN for 3 days; the chondrocytes in vacant group (group C1) were cultured in DMEM medium for 5 days. The expression of Col II and MMP-13 were analyzed by immunocytochemical staining observation and real time RT-PCR test. Another twenty-four 3-month-old male Japanese White rabbits were randomized into three groups (n=8 per group). The OA model was made by ACLT in ACLT+ALN group (group A2) and ACLT group (group B2); the joint cave was sutured after exposure of ACL in sham group (group C2). After 4 days, the rabbits of group A2 received the subcutaneous injection of ALN at a dosage of 10 μg/(kg·d) for 8 weeks. Rabbits of group B2 and C2 received equal normal sal ine treatment. After 8 weeks, the rabbits were executed. The macro-pathologic changes of right knee joints were observed, so were the histological changes of femoral condyles. Expression levels of Col II and MMP-13 were detected by immunohistochemical staining. The bone histomorphometry analysis was appl ied to subchondral bone of proximal tibia. Results In vitro, the Col II immunocytochemical staining showed intensely positive staining in group C1, and the intensity of staining was sl ightly decreased in group A1, but the intensity of Col II immunocytochemical staining was extremely lower in the group B1. The integrated absorbance (IA) value for Col II in group A1 was significantly higher than that of group B1 (P lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). Immunocytochemical detection of MMP-13 showed intense staining in group B1, and the intensity of staining was sl ightly decreased in group A1, but no MMP-13 expression was detected in the group C1. The IA value for MMP-13 in group A1 was significantly lower than that of group B1 (P lt; 0.05), but significantly higher than that of group C1 (P lt; 0.05). The real time RT-PCR analysis showed significantly higher mRNA levels of Col II in group A1 than in group B1 (P lt; 0.05), but there was no significant difference between group A1 and group C1 (P gt; 0.05). The MMP-13 mRNA level of the chondrocytes in group A1 was significantly lower than that of group B1 (P lt; 0.05), but significantly higher than that of group C1 (P lt; 0.05). In vivo, the gross appearance of surface of knee joint showed that there was no ulcer in group C2, and there was some ulcers in group A2, but many and all layers ulcers in group B2. Mankin score of group A2 was significantly lowerthan that of group B2 (P lt; 0.05), but significantly higher than that of group C2 (P lt; 0.05). Immunohistochemical staining showed that Col II in articular cartilage was intensely staining in group C2, the intensity of staining was sl ightly decreased in group A2, and the intensity of Col II immunohistochemical staining was extremely low in group B2, but there was no significant difference between group A2 and group C2 (P gt; 0.05..........
To observe the histology change of the insertion using different diamertrical bone tunnel in anterior cruciate l igament (ACL) reconstruction. Methods Ninety Japanese rabbits were selected, wihout female and male l imit, weighing 2.5-3.0 kg, and were randomly divided into 3 groups, 30 in each group. The ratio of transplantation l igament diameter and bone tunnel diameter was 1/1 (group A), the ratio was 1/1.5 (group B), and the ratio was 1/2 (group C). Bone tunnel observation and histology observation were carried out in the 4th, 8th and 16th weeks postoperat ively. Results Wound healed well in 3 groups. The mean time of walking functional recovery was 1.5, 2.0 and 3.5 days in groups A, B and C respectively. After 4 weeks of operation, more soft tissues at tunnel entry were observed in group A and group B than in group C; after 8 weeks of operation, there was no crevice at bone-tunnel entry of the groups A and B, there was no improvement in group C; after 16 weeks of operation, groups A and B showed the normal insertion, group C had no normal insertion. Histology observation: in groups A, B and C, bone-tunnel was filled with loose connective tissue after 4 weeks of operation; group A and group B emerged the discontinuation ACL insertion tidal l ine after 8 weeks of operation, group C had no insertion; groups A and B emerged the similarity normal ACL insertion tidal l ine structure after 16 weeks of operation, but group C had no this structure. The results of ultimate tensile strength in groups A, B and C were (75.44 ± 7.06), (91.37 ± 6.14) and (126.91 ± 4.61) N respectively at 4 weeks; the results were (74.31 ± 4.81), (88.30 ± 7.46) and (124.34±8.44) N respectively at 8 weeks; and the results were (62.20 ± 5.32), (71.53 ± 5.99) and (83.62 ± 5.69) N respectively at 16 weeks. There was no significant difference between group A and group B (P gt; 0.05), and there were significant differences between groups A, B and group C (P lt; 0.05). Conclusion In the ACL reconstruction, the ratioof transplantation l igament diameter and bone tunnel diameter being 1/1.5 will not affect the insertion outcome, but if theratio less than the l imit it will affect the insertion outcome.
Objective To study the effect of various doses of estrogen on tissue injury, blood supply and survival area of skin flap and to investigate its mechanism. Methods Thirty New Zealand white rabbits aged 3-4 months old and weighing 1.5-2.2 kg (male or female) were used. Random pattern skin flap (12 cm × 3 cm in size) taking the central l ine of the rabbit dorsum as axis and with the pedicle attached at the proximal end was prepared, and the flap pedicle division was performed 7 days after operation. The rabbits were divided randomly into three groups (n=10 rabbits per group). At 2, 4, and 6 days after operation, the proximal edge of flap in group A and B received 100 ?g/kg and 50 ?g/kg subcutaneous injection ofestradiol benzoate, respectively, while group C received no further treatment serving as control group. General condition ofthe rabbits was observed after injection, gross observation was performed 3 and 7 days after injection, survival area of the skin flap was measured 7 days after injection, contents of malondialdehyde (MDA) and nitric oxide (NO) were tested 5 days after injection, and the flaps were harvested 4 and 7 days after injection to receive histology and no significant difference was noted between group A and group B (P gt; 0.05). The NEU counts 4 days after injection were (18.20 ±6.24) cells/HP in group A, (21.27 ± 5.34) cells/HP in group B, and (28.78 ± 7.92) cells/HP in group C, and at 7 days after injection, there were (15.16 ± 7.02) cells/HP in group A, (18.12 ± 6.44) cells/HP in group B, and (29.67 ± 9.12) cells/HP in group C. The VEGF score 4 days after injection was (4.02 ± 0.48) points in group A, (4.19 ± 0.66) points in group B and (3.67 ± 0.49) points in group C, and at 7 day after injection, it was (4.96 ± 0.69) points in group A, (5.12 ± 0.77) points in group B, and (3.81 ± 0.54) points in group C. Significant difference was evident between 4 days and 7 days after injection in group A or B in terms of NEU counts and VEGF score (P lt; 0.05), and difference between 4 days and 7 days after injection in group C was not significant (P gt; 0.05), and the differences among 3 groups were significant (P lt; 0.05). Conclusion Estrogen injection can increase VEGF expression and NO content of flap, decrease MDA content and NEU infiltration of flat, and improve survival area of flap.
Objective To observe effects of the core binding factor α1 (Cbfα1) in its promoting differentiation of the rabbit marrow mesenchym al stem cells (MSCs) into osteoblasts. Methods The rabbit marrow MSCs were isolated and cult ured in vitro and were divided into 3 groups. In the control group, the marr ow MSCs were cultured by DMEM; in the single inducement group, they were cultured by the condition medium (DMEM, 10% fetal bovine serum, dexamethasone 10 mmol/L, vitamin C 50 mg/L, and βGP 10 mmol/L); and in the experimental group , the ywere transfected with AdEasy1/Cbfα1,and then were cultured by the condition m edium. The alkaline phosphatase(ALP) activity and the experission of osteocalcin as the osteoblast markers were measured with the chemohistological and immunohi stochemical methods at 3 days，1，2，3，and 4 weeks after inducement. Results More than 90% MSCs were grown well in vitro. The GFP was positive in MSCs after their being transfectived with AdEasy1/Cbfα1. The ALP activity and the experission of osteocalcin were significantly upregulated in the transfection group compared with those in the single inducement group and the control group at 1, 2, 3, and 4 weeks （Plt;0.05）.The mineralized node began to appear at 2 weeks in the experiment al group and the single induction group, but did not appear in control group. Conclusion Cbfα1 can obviously promote differentiation of the rabb it marrow mesenchymal stem cells into the osteoblasts.