Objective To observe the effect of pigment epithelium-derived factor (PEDF）on glutamate metabolism in diabetic rat retina. Methods 78 Sprague-Dawley rats were randomly divided into the model group, model control group, PEDF intervention group and intervention control group. There were some dead and euglycemia rats at the end of experiment, so only 12 rats in each group were included in the statistical analysis. The diabetic retinopathy rat model of the model, PEDF intervention and intervention control group were induced with streptozotocin injection. The rats in the model group were not intervened. The monthly-age matched normal rats of model group were in the model control group. The left eyes of rats were received intravitreal injection with 5 mu;l (0.1 mu;g/mu;l) PEDF (PEDF intervention group) or 5 mu;l phosphate buffer solution (intervention control group). The expressions of L-glutamate/L-aspartate transporter in retina were analyzed by western blot and real time RT-PCR techniques and glutamate content in retina was analyzed by high-pressure liquid chromatography (HPLC). Cultured rat Muuml;ller cells were divided into the control,experimental, PEDF intervention and intervention control group, GLAST expressions were detected by fluorescence immunofluorescence and real-time RT-PCR techniques. The glutamate up-take activity of Muuml;ller cells was determined by intracellular [3H] labeled D, L-glutamate concentration with scintillation counting. Results Western blot and real-time RT-PCR showed that GLAST expression decreased (real-time RT-PCR：t=8.86,Plt;0.01；Western blot：t=3.42,P＜0.05), glutamate content increased（t=4.01,P＜0.05）in model group compared with the model control group; GLAST expression increased (real-time RT-PCR：t=3.56,P＜0.05；Western blot：t=3.52,P＜0.05), glutamate content decreased（t=4.36,P＜0.05）in the PEDF intervention group compared with the intervention control group. Real-time RT-PCR and fluorescence immunofluorescence showed that high glucose down-regulate GLAST expressions in Muuml;ller cells (rea-time RT-PCR：t=3.48,P＜0.05；fluorescence immunofluorescence：t=4.72,P＜0.05 ) and impair glutamate uptake activity of Muuml;ller cells (t=3.81, Plt;0.05). Under high glucose conditions, PEDF up-regulated GLAST expression significantly (real-time RT-PCR：t=6.82,P＜0.01；fluorescence immunofluorescence：t=3.72,P＜0.05) and ameliorated the glutamate up-take activity of Muuml;ller cells（t=4.14, Plt;0.05). Conclusions In diabetic rats, PEDF may improve the activity of GLAST in Muuml;ller cells, thus ameliorate retinal glutamate metabolism and inhibit death of retinal ganglion cells.
Objective To investigate the effect of pigment epitheliumderived factor (PEDF）on the expression of glutamine synthetase in retinal Muuml;ller cells of diabetic rats.Methods Diabetic rats were induced with streptozotocin injection.Before and after injection of 10 mu;l (0.1 mu;g/mu;l) PEDF (experimental group) or 10 mu;l PBS (control group) into the vitreous cavities of diabetic rats respectively for 48 hours,the expressions of GS and IL-1beta; in retina were analyzed by immunohistochemistry and real time RTPCR techniques. After being treated with 100 ng/ml PEDF for 24 hours in high glucose conditions,the expressions of GS and IL-1beta; in cultured Muuml;ller cells were studied by western blot and real time RT-PCR techniques. Apoptosis was analyzed by flow cytometry after Annexin V fluorescein isothiocyanate/Propidium idoium (Annexin V-FITC/PI) staining.Results By immunohistochemistry (the protein level) and real time RT-PCR (the mRNA level),it was found that the expression of GS decreased and the expression of IL-1beta; increased obviously (real time RT-PCR：GS:t=4.23,P＜0.01；IL-1beta;:t=16.73,P＜0.01；immunohistochemistry：GS: t=5.13,P＜0.01；IL-1beta;:t=9.32,P＜0.01) in diabetic rats. After injection of 10 mu;l (0.1 mu;g/mu;l) PEDF into the vitreous cavities of diabetic rats for 48 hours,it was found that the expression of GS increased and the expression of IL-1beta; decreased significantly(RT-PCR GS:t=3.87,P＜0.01IL-1beta;:t=3.61,P＜0.05;immunohistochemistry:GS:t=3.32, P＜0.05；IL-1beta;: t=2.63,P＜0.05). Under high glucose conditions, 100 ng/ml PEDF induced decreasing expression of IL-1beta; and increasing expression of GS significantly (RT-PCR：GS: t=2.89, P＜0.05；IL-1beta;: t=3.37,P＜0.05；Western blot：GS:t=2.66,P＜0.05;IL-1beta;:t=3.23,P＜0.05).Apoptosis of Muuml;ller cells under high glucose conditions was inhibited significantly by the treatment with 100 nmol/ml PEDF (t=3.21,P＜0.05). Conclusions In diabetic rats,PEDF may decrease expression of IL-1beta; in rat retinal Muuml;ller cells, which may result in increasing expression of GS.To some degree,it inhibits possibly the death of retinal ganglion cells.
Objective To investigate the feasibility of establishing an anterior cruciate ligament (ACL) reconstruction model using hamstring tendon autograft in cynomolgus monkeys. Methods Twelve healthy adult male cynomolgus monkeys, weighing 8-13 kg, were randomly divided into two groups (n=6). In the experimental group, the ACL reconstruction model of the right lower limb was prepared by using a single bundle of hamstring tendon, and the ACL of the right lower limb was only cut off in the control group. The survival of animals in the two groups was observed after operation. Before operation and at 3, 6, and 12 months after operation, the knee range of motion, thigh circumference, and calf circumference of the two groups were measured; the anterior tibial translation D-value (ATTD) was measured by Ligs joint ligament digital body examination instrument under the loads of 13-20 N, respectively. At the same time, the experimental group underwent MRI examination to observe the graft morphology and the signal/ noise quotient (SNQ) was caculated. Results All animals survived to the end of the experiment. In the experimental group, the knee range of motion, thigh circumference, and calf circumference decreased first and then gradually increased after operation; the above indexes were significantly lower at 3 and 6 months after operation than before operation (P<0.05), and no significant difference was found between pre-operation and 12 months after operation (P>0.05). In the control group, there was no significant change in knee range of motion after operation, showing no significant difference between pre- and post-operation (P>0.05), but the thigh circumference and calf circumference gradually significantly decreased with time (P<0.05), and the difference was significant when compared with those before operation (P<0.05). At 6 and 12 months after operation, the thigh circumference and calf circumference were significantly larger in the experimental group than in the control group (P<0.05). At 3 and 6 months after operation, the knee range of motion was significantly smaller in the experimental group than in the control group (P<0.05). Under the loading condition of 13-20 N, the ATTD in the experimental group increased first and then decreased after operation; and the ATTD significantly increased at 3, 6 months after operation when compared with the value before operation (P<0.05). But there was no significant difference between the pre-operation and 12 months after operation (P>0.05). There was no significant change in ATTD in the control group at 3, 6, and 12 months after operation (P>0.05), and which were significantly higher than those before operation (P<0.05). At each time point after operation, the ATTD was significantly smaller in the experimental group than in the control group under the same load (P<0.05). The MRI examination of the experimental group showed that the ACL boundary gradually became clear after reconstruction and was covered by the synovial membrane. The SNQ at each time point after operation was significantly higher than that before operation, but gradually decreased with time, and the differences between time points were significant (P<0.05). Conclusion The ACL reconstruction model in cynomolgus monkey with autogenous hamstring tendon transplantation was successfully established.