The damage effects of the pure tumor necrosis factor (TNF) on the normal animals were observed. Eighteeen rabbits were divided into two groups, eight in tested group and ten in control group. 0.5mg per kg of the pure rabbit TNF was given to each animal of the tested group. Results:The symptoms similar to that induced by endotoxin appeared after the TNF injection. The functions of the main organs were markedly damaged. The arterial blood pressure of most animal was low. The weight ratio of the orgen to the body was raised. The pathologic changes were similar to those of the multiple organ failure (MOF) model. Most of the animal died before the end of the experiment. The results suggest that pure TNF could indece multiple organ damages similar to those of MOF.
Objective To observe the effects of immunologic cytokines or anti-angiogenesis gene transfer mediated by electroporation for choroidal melanoma cells.Methods The human embryo kidney cells and malignant choroidal melanoma cells were transfected with plasmids pNGVL-mIL2, pNGVL-mIL12, pCI-sFLK-1, pCR3.1-antiVEGF121,pCI-ExTek. Then the expression of mIL2, mIL12, sFLK-1, VEGF and ExTek were detected by enzymelinked immunosorbentassay (ELISA) and Western blot. Nude mice models of malignant choroidal melanoma were established and they were divided into four groups randomly. Each group was treated with 30 mu;l of 0.9% NaCl, 30 mu;g pNGVL, 30 mu;g antiVEGF121+sFLK-1+ExTek and 30 mu;g mIL2+mIL12 respectively by electroporation. Seven，14, 21, 28, 35 and 42 days after treatment, the tumor volumes were measured to calculate the tumor inhibition rate. Results ELISA and Western blot showed that mIL2，mIL12，sFLK-1 and ExTek were expressed after electroporation，VEGF expression was decreased remarkably. After treatment，the tumors of mIL2+mIL12 group were greatly inhibited with a tumor inhibition rate of 97.33%，while the tumors of antiVEGF121+sFLK-1+ExTek and pNGVL group were partially inhibited with tumor inhibition rates of 53.33% and 36.33% respectively.Conclusions Immunologic cytokines transfer mediated by electroporation can inhibit the growth of melanoma，but anti-angiogenesis only have a mild effects.
Objective To investigate if lactic acid can promote the expression of vascular endothelial growth factor (VEGF) in the rat retinal explants.Methods The retinas of two-week neonatal SD rats were placed onto the culture plate inserts and incubated with Dulbeccoprime;s modified Eagleprime;s medium （DMEM） plus 2% fetal bovine serum (FBS) containing 10,20,30 mmol/L of lactic acid, respectively. Each group had 24 retinas. At 24 hours after incubation, the retinas were sectioned for light microscopy and the expression of VEGF was measured by real time PCR and Western blot. Results The cultured retinas maintained intact construction, and no cytolysis and apoptosis were observed under light microscope. RT-PCR showed the levels of VEGF mRNA were 0.74plusmn;0.06 for 10 mmol/L lactic acid group, 0.99plusmn;0.12 for 20 mmol/L group, and 1.45plusmn;0.17 for 30 mmol/L group respectively. VEGF expression was 0.34plusmn;0.15 for 10 mmol/L, 0.54plusmn;0.16 for 20 mmol/L, and 0.93plusmn;0.23 for 30 mmol/L group respectively by Western blot. Both PCR and Western blot showed 30 mmol/L of lactic acid significantly increased the levels of VEGF mRNA and VEGF expression. Conclusion The induction of retinal VEGF by lactic acid is concentration-dependent.
Objective To investigate the inhibitory effects of 15-lipoxygenase-1 (15-LOX-1) gene transfer on oxygen-induced retinal neovascularization in mice. Methods Ninety-six 7-day-old C57BL/6J mice were randomly divided into normal control group, oxygeninduced retinopathy (OIR) model group, gene treated group and empty vector group. The mice with their mothers were kept in (75plusmn;2) % 02 environment for 5 days and then returned to normoxia for 5 days to establish the OIR model. At postnatal day 12, the gene treated group received intravitreous injection of recombinant adenovirus (Ad) vector containing both enhanced green fluorescent protein (EGFP) and mouse 15-LOX-1 genes (Ad-15-LOX-1-EGFP) at 1 l, while the empty vector group received the same volume of recombinant Ad vector containing EGFP (Ad-EGFP). The expression of EGFP was observed on flat-mounted retina by fluorescence microscopy 2 days after intravitreous injection of Ad-15-LOX-1-EGFP. At postnatal day 17, the efficacy of 15-LOX-1 gene transfer on retinal tissue was detected by immunofluorescence staining, real-time polymerase chain reaction and Western blot. The changes of retinal vessels, relative retinal non-perfusion and neovascularization areas were evaluated by fluorescein isothiocyanate-dextran fluorescein angiography on flatmounted retina. The number of endothelium cell nuclei breaking through the inner limiting membrane (ILM) was counted on hematoxylin and eosin-stained retinal section. Results Two days after intravitreous injection of Ad-15-LOX-1-EGFP, the expression of EGFP had been seen by fluorescence microscopy on Flat-mounted retina. Immunofluorescence staining of retinal section revealed that 15-LOX-1 expression was primarily in the outer plexiform layer, inner nuclear layer and ganglion cell layer of retina. The 15-LOX-1 protein and mRNA expression levels were higher in gene treated group than those in OIR model group and empty vector group (tprotein=22.74 and 24.13 respectively.tmRNA=12.51 and 13.40 respectively; P＜0.01). The relative retinal non-perfusion and neovascularization areas were significantly smaller in gene treated group than those in OIR model group and empty vector group (tnon-perfusion=16.22 and 14.31 respectively.tneovascularization=9.97 and 9.07 respectively; P＜0.01), and the number of endothelium cell nuclei breaking through the ILM in gene treated group was obviously lower than the other two groups (t=14.25 and 11.62 respectively，P＜0.01). Conclusion 15-LOX-1 gene transfer can decrease the oxygen-induced retinal non-perfusion areas and inhibit the retinal neovascularization in mice.
Standard venographies were pcrformed to evaluate the endothelial damage by the contrast medium. After different time intervals, the local veins were prepared for transmission and scanning electron microscopy (TEM and SEM) investigation. The veins were in a dilated state after the angiographies, which lasted for about two days. The endothelial damage was most severe 1 day after the venography. Besides the lesions of extensive endothelial tissurs, dcsquamations, and the exposure of subendothelial tissues, microthrombi somethimes were found. Healing occurred within 3 days. The results this study has also verifieed that it was more valuable to study venogqaphic effects on veins with TEM and SEM.
Objective：To observe the expression of gene and protein l evel of unfolded protein, glucoseregulated protein 78 (GRP78), after retinal d etachment (RD); to find out the relationship between UPR and the cell damage after RD. Methods：Eightyeight Wistar rats were divided into 2 groups: con trol group (11 rats) and RD group (77 rats). In RD group, subretinal injection with 10 mg/ml hyaluronic acid sodium was performed on the left eyes of the rats t o set up RD model, and the left eyes and retinal tissue were collected 1/2 day, 1 day, 2, 4, 8, 1 6 and 32 days after RD; there were 11 rats in each subgroup. The expression of G RP78 mRNA in retina tissue was detected by semiquantitative reverse transcript i on polymerase chain reaction (RT－PCR), the expression of GRP78 protein level wa s detected by Western blotting, and the distribution of GRP78 in each retinal lay er was observed by immunofluorescence labeling method and confocal microscopy. Results：The expression of retinal GRP78 mRNA significantly in creased in 1/2 day , 1 day, 2, and 4 days subgroups after RD (Plt;0.05). The expression of GRP7 8 protein significantly increased in each subgroup after RD compared with which in the control group, and reached the peak in 8, 16, and 32 days subgroups. The expres sion of GRP78 protein was detected in all of the retinal layers after RD. Conclusion：The protection mechanism of UPR starts up after RD, and l eads the correc t pucker of the protein and reduces cellular injury by upregulating the expres s ion of GRP78, which provide the theoretic basis for reducing the cellular injury and improving the visual function in patients with RD.
Objective：To observe the protective effect of ginkgo bilo ba extrac t (EGb 761), a free radical scavenger, on the photoreceptor cells after lighti nduced retinal damage. Methods：Seventytwo female SpragueDa wley (SD) rats we re randomly divided into 4 groups: normal control group, lightinduced retinal da m age model group, model+physiological saline group, and model+EGb 761 group, with 18 rats in each group. All of the rats except the ones in the control group were exposed to white light at (2740plusmn;120) lx for 6 hours after the dark adap tation for 24 hours to set up the lightinduced retinal damage model. Rats in m o del + physiological saline group and model+EGb 761 group were intraperitoneall y injected daily with physiological saline and 0.35% EGb 761 (100 mg/kg), respec tively 7 days before and 14 days after the light exposure. Apoptosis of photorec eptor cells was detected 4 days after light exposure; 7 and 14 days after light exposure, histopathological examination was performed and the layer number of ou ter nuclear layers (ONL) on the superior and inferior retina was counted. Results：Four days after light exposure, the apoptosis of photorecep tor cells was fou nd on ONL in model, model+ physiological saline and model+EGb 761 group, and w as obviously less in model + EGb 761 group than in model and model+physiologic al saline group. Seven days after light exposure, the layers of ONL on the super ior retina were 3 to 4 in model and model+physiological saline group, and 7 to 8 in model+EGb 761 group; the mean of the layer number of ONL in model+EGb 761 group (6.92plusmn;0.82) was less than that in normal control group (8.40plusmn;0.95) (t=-1.416, P＜0.05), but significantly more than that in model (5.96 plusmn;1.36 ) and model+physiological saline group (5.90plusmn;1.40）（t=1.024, 1.084; P＜0.05). Fourteen days after light exposure, the layers of ONL on the superior retina were 0 to 1 in model and model+physiological saline group, and 3 to 4 i n model+EGb 761 group. The mean of the layer number of ONL in model+EGb 761 group (5.5 2plusmn;1.06) was significantly more than that in model (3.44plusmn;2.15) and model + physiological saline group (3.37plusmn;1.91) (t=2.082, 2.146, P＜0.05). Conclusion：EGb 761 can partially inhibit the apoptosis of pho toreceptor cells, thus exert protective effect on photoreceptor cells.
ObjectiveTo investigate the influence of Ataxia-telangiectasia mutated (ATM) activation on cellular oxidative stress induced by high glucose in bovine retinal capillary endothelial cells(BRECs). Methods The BRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 30 mmol/L glucose, 30 mmol/L glucose+10 μmol/L KU55933) as normal glucose group, high glucose group and treatment group respectively.After the cells incubated for 48 hours, the protein expression of ATM, P-ATM, Mitogen-Activated Protein Kinase P38(P38), P-P38, Extracellular signal-regulated kinases(ERKs), P-ERKs was detected by Western blot; cellular ROS level was detected by Reactive Oxygen Species Assay Kit; propidium iodide/Hoechst staining was used for analysis of apoptosis; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by Enzyme-Linked Immunosorbent Assay (ELISA); the paracellular permeability between endothelium cells was detected by FITC-dextran. ResultsCompared with the protein level of P-ATM, P-P38 and P-ERKs in high glucose group increased. Especially, P-P38, P-ERKs expressed much more than in high glucose group. The secretion of VEGF in high glucose group was higher than that in the normal glucose group but less than that in treatment group. The same tendency existed in ROS assay, apoptosis assay and paracellular permeability measuring. ConclusionsHigh glucose induced altered activation of ATM which might play a protective role in cellular oxidative stress. Deficiency of ATM might lead to ROS explosion, cell apoptosis and dysfunction of endothelial barrier. The mechanism might be associated with P38, ERKs and VEGF.
Objective To observe the characteristics of the full-field flash electroretinogram (F-ERG) in rats with oxygen induced retinopathy (OIR). Methods Twenty-four neonatal Sprague Dawley rats were divided into OIR group and control group. In OIR group, 12 rats were exposed to (75±2)% oxygen for 7 days and then to room air for 7 days; in control group, 12 rats were raised in room air for 14 days. At postnatal day 21, F-ERG tests were performed to examine the rod response , the maximum mixing reaction and the cone reaction. Results Compared with the control group, the b-wave amplitudes decreased (t=3.650) and the implicit times increased (t=2.410) in rod response in OIR group, the differences were statistically significant (P＜0.05); the a- and b-wave amplitudes decreased (t=3.333, 2.562) and the implicit times increased (t=2.725, 2.482) in the maximum mixing reaction in OIR group, the differences were statistically significant (P＜0.05). There was no difference between OIR and control group on a- and b-wave amplitudes (t=0.650, 0.204) and implicit times (t=0.422, 0.076) in cone response (P＞0.05). 0.001 cd.s/m2 light intensity stimulation on rats F-ERG wave almost no response. 0.010 cd.s/m2 light intensity stimulation on rats can be recorded to the rod response waveform, with the increase of light intensity, the amplitude of b-wave increases, the a-wave extraction. Conclusions F-ERG of OIR rat showed that the amplitude and sensitivity of the rod response and maximal rod-cone response was decreased. The intensity of light had effect on the OIR rod cells, and the amplitude of b- wave increased with the increase of light intensity, the a-wave extraction.