Objective To study the effect of splenectomy on the anti-tumor immunity in rats with induced hepatocellular carcinoma (HCC). Methods At the second and fourth month of the induced HCC, the NK cell activity, TNF-α level and total lymphcyte in blood were measured in the group of splenectomy and the control group. Results There were no different in the total lymphcyte and TNF-α in the blood in two groups, but there were significant difference in the NK cell activity between the group of splenectomy and the control group (Ｐ＜0.05). Conclusion There are some change in the anti-tumor immunity after splenectomy in rats, in which NK cell activity is at low level continuously. TNF-α isn′t affected after the second month after splenectomy.
ObjectiveTo observe the expression of interleukin (IL)-17, IL-4 and interferon γ (IFN-γ) in experimental autoimmune uveoretinitis (EAU). MethodsC57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein 1-20 to induce EAU. The inflammatory reaction before and on 7, 14, 21, 28 days after immunization were observed. The level of IL-17, IL-4 and IFN-γ in the serum were measured by enzyme-linked immune sorbent assay (ELISA). mRNA and protein expression of spleen and retina were analysed using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot at the same time, respectively. ResultsThe most serious inflammatory reaction occurred at the 14th day after immunization. The highest level of IFN-γ in serum, highest mRNA and protein expression of IFN-γ in spleen and retina of mice occurred at day 7 after being immunized. The highest level of IL-17, IL-4 in serum, highest mRNA and protein expression of IL-17, IL-4 in spleen and retina of mice occurred at day 14 after being immunized. The increase degree of IL-17 was more than IFN-γ and IL-4. At 7, 14 and 21 days after immunization, compared with the pre-immunization, the level of IL-17, IL-4, IFN-γ in serum of mice were significantly increased (F=1 817.346, 268.600, 164.621; P < 0.05). There was no difference in the levels of IL-17, IL-4, IFN-γin serum of mice between pre-and 28 days after immunization (P > 0.05). At 7, 14 and 21 days after immunization, compared with the pre-immunization, the protein expression of IL-17, IL-4, IFN-γ in spleen (F=312.67, 114.250, 216.220) and retina (F=271.504, 85.370, 80.722) of mice were significantly increased (P < 0.05). There was no difference in protein expression of IL-17, IL-4, IFN-γ in spleen and retina of mice between pre-and 28 days after immunization (P > 0.05). ConclusionsThere were IL-17, IL-4 and IFN-γ expression in EAU. IL-17, IL-4 and IFN-γ play a key role in the occurrence and development of the EAU.
Objective:To observe the effect of beta;estradiol on gluta mate concentration in rabbitsprime; retinae injured by ischemic reperfusion. Methods:Twenty r abbits ware randomly divided into two groups, the control group and the treatmen t group, with 10 rabbits in each group. Before examined by binocular flash elect roretinography (FERG), retinal ischemic reperfusion (RIR) model was induced in t h e right eyes of all the rabbits by increasing intraocular pressure to 120 mm Hg for 60 minutes; the left eyes were as the control eyes. The rabbits were hypoder mically injected with beta;estradiol (0.1 mg/kg) in treatment group and with phys i ological saline in the control group 2 hours before ischemia. The results of FER G of the right eyes in both of the 2 groups 0, 4, 8, and 24 hours after reperfus ion were record respectively and were compared with the results of FERG before r eperfusion. The retina tissue was collected after the last time of FERG. The con c entration of glutamate was detected by Hitachi L8800 amino acid analyzer. Results:In the right eyes in both of the 2 groups, the result of F ERG showed a beeli ne just after reperfusion. There was no significant difference of awave amplit u de between the 2 groups (t=1.357, 0.798, 0.835; Pgt;0.05); the b wave amplitudes i n experimental group were much higher than those in the control group (t=4.447, 2.188, 3.106; Plt;0.01). The concentration of glutamate in retina was (0.265plusmn;0.014) g/L in the right eyes and (0.207plusmn;0.013) g/L in the left eyes in the control group, and (0.231plusmn;0.007) g/L in the right eyes and (0.203plusmn;0 .014) g/L in the le ft eyes in the treatment group; the difference between the 2 groups was signific ant (F=50.807, P=0.000). There was statistical difference between righ t and left eyes both in the 2 groups and the significant difference of the right eyes betw een the two groups was also found (P=0.000); there was no statistical diffe rence of the left eyes between the 2 groups (P＝0.505). Conclusion:beta;-estradiol may prevent the increase of the concentration of glutamate in retina induced by RIR to protect retinal tissue.
Objective To investigate the effects of lights with different wavelength on the retina of rd12 and C57BL/6J mice. Methods Thirty two rd12 mice and C57BL/6J mice were randomly divided into the control group, white light group, midwavelength light (505 nm) group and shortwavelength light (405 nm) group, with eight mice in each group. Besides the control group, other groups were exposed to cycle illuminations ［12 hours dark, 12 hours (800plusmn;130) Lux］ for seven days to establish the model of retinal light damage. Electroretinogram (ERG) responses of all mice were recorded at the day before illumination and 1st, 4th and 7th days after illumination. The eyes were enucleated at 7th days after illumination to assess levels of reactive oxygen species (ROS), expression of peroxiredoxin 6 (PRDX6), and activity of caspase-3. Results ERG amplitudes of all groups declined gradually in C57BL/6J mice, and the most significant effects was found in the short-wavelength light group. The amplitudes of photopic b-wave were significantly different at 1st, 4th and 7th days (F=4.412, 5.082, 9.980；P＜0.01). The amplitudes of cone b-wave of the four groups decreased to (85plusmn;10) %, (70plusmn;19) %, (57plusmn;22) % and (46plusmn;19) % at 7th days, respectively, and were significantly different between white light group and short-wavelength light group(t=3.19,P＜0.01). The levels of ROS were significantly different in rd12 mice (F=16.08,P＜0.01), and elevated obviously in shortwavelength light group. The expressions of PRDX6 of retina were significantly different in rd12 mice (F=7.214,P＜0.05), and were decreased obviously in short-wavelength light group. The caspase-3 relative activity was significantly different in rd12 retina (F=7.530,P＜0.05); but there was no significant difference in C57BL/6J mice (F=3.625, 1.993, 1.133; P＞0.05).The caspase-3 relative activity were significant different between rd12 mice and C57BL/6J mice in short wavelength light group (t=5.474,P＜0.05). Conclusions Short-wavelength light can induce retinal damage of mouse retina, especially in rd12 mouse. The retinal light damage possibly relates to the oxidative damage.
Objective To establish a rat model of blood ocular barrier breakdown induced by anterior segment intraocular analogic surgery. MethodsOne hundred and fifty healthy adult male rats were randomly divided into control group and model group, 75 rats in each group. The rats were anesthetized with 1 ml/kg ketamine hydrochloride/xylazine hydrochloride solution. Three way pipes were attached to a phosphate buffer infusion bag and two intravenous catheters. One catheter was inserted 30° obliquely through the transparent cornea anterior to the limbus into the rat's anterior chamber. Then the needle was withdrawn and the sheath was indwelling. Another catheter was connected with a manometer. Intraocular pressure was varied from 0 to 12 mm Hg (1 mm Hg=0.133 kPa) 60 times, 30 times per min. The catheter was removed. The eyes were treated with ofloxacin ophthalmic solution after surgery. The 1st, 2nd, 3rd, 5th and 7th day after surgery, the integrity of the blood ocular barrier was assessed by immunohistochemical staining for albumin and quantitative measurement using Evan′s blue as a tracer. ResultsAlbumin immunohistochemical staining of the control group was confined to the iris and retinal blood vessels. The choroid was stained at each time point after surgery. Albumin immunohistochemical staining of the model group was abundant around the iris and the retinal vasculature on the 1st day after surgery. The albumin diffused throughout the iris and the retina on the 2nd and the 3rd day after surgery. The albumin reached the retinal vessels on the 5th and 7th day after surgery. The aqueous humor Evans blue leakages of the model group were higher than those of the control group on the 1st, 2nd, 3rd and 5th day after surgery. The differences were statistically significant (t=25.781, 37.433, 25.150, 19.171； P<0.01). The Evans blue leakage of the model group was close to that of the control group on the 7th day after surgery. The difference was no statistical significant（t=1.303, P=0.209）. The retinal Evans blue leakages of the model group were higher than those of the control group on the 1st, the 2nd and the 3rd day after surgery. The differences were statistically significant (t=11.997, 14.622, 23.014; P<0.01). The Evans blue leakage of the model group was close to those of the control group on the 5th and 7th day after surgery. The differences were not statistically significant（t=2.027, 0.756; P=0.058, 0.459）. Conclusion This study establishes a rat model of blood ocular barrier breakdown induced by imitating the injury to the anterior segment during intraocular surgery.
ObjectiveTo explore the inhibition effect of Cysteine-rich 61(CCN1;Cyr61) specific siRNA expression vector on RNV in a mouse model of oxygen-induced retinopathy (OIR). MethodsOne hundred and twenty healthy C57BL/6J mice were chosen and randomly divided into the experimental group and control group, with 60 mice in each group. The experimental group was intravitreously injected with CCN1siRNA recombinant plasmids. The control group was injected with vector plasmids. Adenosine diphosphate-ase stained retina flat-mounts was performed to assess the retinal vascular profiles, retinal section with HE staining was applied to count the number of new vascular cell nuclei and the protein and mRNA expression of CCN1 and vascular endothelial growth factor (VEGF) were detected by immunohistochemistry, Western blot and Real-time RT-PCR. ResultsCompared with control group, regular distributions, good branches and reduced density of retinal neovascularization were observed in the experimental group. The number of nucleus of vascular endothelial cells breaking through the inner limiting membrane was obviously less in the experimental group than that in the control group (t=8.756, P < 0.05). The expression of CCN1 and VEGF were obviously decreased in the experimental group compared with the control group (all P < 0.05). ConclusionThe development of RNV of ROP can be markedly inhibited by RNA interference targeting CCN1, and CCN1siRNA may provide an effective method for preventing vascular proliferative retinopathy.
ObjectiveTo observe the expression of Caspase-3, Caspase-8, Bcl-2, Bax in the rat retina of ocular ischemic syndrome (OIS). Methods30 Brown Norway rats were randomly divided into experimental group and control group, 15 rats in each group. The rats in experimental group were established a model through ligating the bilateral common carotid artery. At 3 months after modeling, the retinal thickness and ganglion cell (RGC) density were measured by hematoxylin eosin staining; the expression of Caspase 3, Caspase 8, Bax and Bcl-2 in the retina was measured by quantitative real-time reverse transcription polymerase chain reaction. ResultsThe RGC density in control group and experimental group was 61.97±9.07 and 38.1±5.98, respectively. Compared to the control group, the RGC density was diminished in the experimental group (t=3.059, P < 0.01). A significant decrease was found in the total retina (t=3.036), inner plexiform (t=3.715), inner nuclear (t=3.339) and outer plexiform (t=3.341) thickness (P < 0.05). However, no change of the thickness was evident in the outer nuclear layers (t=2.000, P > 0.05). The levels of protein and RNA expression of Caspase 3, Caspase 8 and Bax in the retina were increased in experimental group (F=17.036, 7.787, 11.431, 11.162, 17.763, 12.183; P < 0.05), while the Bcl-2 expression were decreased (F=10.298, 12.047; P < 0.05). ConclusionsThere is obvious expression of apoptosis-associated proteins in the rat retina of OIS. Caspase 3, Caspase 8 and Bax expression are increased, while Bcl-2 expression are decreased.
ObjectiveTo study the inhibitory effects of pigment epithelium derived factor (PEDF) on oxygen-induced retinal neovascularization in mice, and to investigate the possible involvement of interleukin-1β (IL-1β) in the neovascular-inhibitory function of PEDF. Methods A total of 140 postnatal day (P)7 C57BL/6 mice were randomly divided into normal control group, oxygen-induced retinopathy (OIR) model group, PEDF treatment group and PBS treatment control group. All mice except normal control group with their mothers were exposed to (75±2)% oxygen environment for 5 days and then kept in room air for another 5 days to establish the OIR model. Mice in normal control group were kept in room air only. At P12 and P14, respectively, mice in PEDF treatment group received intravitreous injections of 1 μl PEDF (2 μg/μl), while PBS treatment control group received the same volume of PBS (10 mmol/L, pH7.4).All mice were euthanized at P17 and eyes were isolated. The changes of retinal vessels were observed on retinal flat mounts and cryosections by fluorescence microscopy. Retinal specimens were prepared for IL-1β protein and mRNA analysis by Western blot and real time fluorescence quantitative reverse transcription-polymerase chain reaction (Real-time RT-PCR). ResultsChanges of retinal vessels had been viewed by fluorescence microscopy on flat-mounted retina, the relative retinal neovascularization areas were significantly increased in OIR model group compared with normal control group (t=15.02, P < 0.01), and the relative retinal neovascularization areas were obviously smaller in PEDF treatment group than those in PBS treatment control group (t=5.96, P < 0.01). Fluorescence staining revealed that retinal vascular tufts were extending from outer plexiform layer (OPL) to ganglion cell layer (GCL) of the retina along with multiple interconnections; Neovascular tufts in OIR model group and PBS treatment control group were presenting distinctly more than those of normal control group and PEDF treatment group. The specific expression levels of IL-1β protein in retinas of OIR mice by Western-blot analysis were higher than those of normal control group(t=3.35, P < 0.05), While these of PEDF treatment group showed a considerable decline in comparison with PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.764, P > 0.05). Similarly, expression levels of IL-1β mRNA tested by Real-time RT-PCR were obviously increased in the OIR model group when compared to normal control group(t=4.43, P < 0.01). After treated with PEDF, expression levels of IL-1β mRNA showed a considerable decrease when compared to PBS treatment control group (P < 0.01), and there were no difference in normal control group and PEDF-treated group (F=11.15, P > 0.05). ConclusionsPEDF can inhibit oxygen-induced retinal neovascularization. The mechanism may be related to that PEDF can downregulate the expression of IL-1β in retina.
Objective To observe the inhibitory effect of recombined adenovirus of p21 (rAd-p21) on the proliferation of Rhesus retinal vascular endothelial cell line (RF/6A). Methods RF/6A cells were cultured in vitro which divided into phosphate buffered solution (PBS) group, rAd-p21 transfection group and negative control (NC) group. Plasmid vectors were transfected into RF/6A cells. The expression of p21 mRNA and protein in RF/6A cells were measured by RT-PCR and Western blot respectively. The cell cycle distribution was analyzed by flow cytometry. Endothelial-cell tube formation assay was performed in Matrigel.Results The expression of p21 mRNA and protein in rAd-p21 transfection group were higher than that in PBS and negative control group. The cell cycle distribution showed that the proportion of G0/G1 cells in rAd-p21 transfection group ［(67.45plusmn;11.61) %］ was apparently higher than that in the negative control group ［(41.55plusmn;8.99)%］ and PBS group ［(40.76plusmn;6.66) %］ (F=21.284, P=0.000). The number of endothelialcell tubes in the rAd-p21 transfection group (3.86plusmn;1.21) was apparently less than that in the negative control group (7.62plusmn;2.69) and PBS group (8.25plusmn;3.19) (F=7.138，P=0.004). Conclusions The p21 mRNA and protein can stably express in RF/6A cells after rAd-p21 transfection. RAd-p21 can inhibit the proliferation of RF/6A cells.
Objective To evaluate the inhibited effects of small interfering RNA targeting Rac1 (Rac1-siRNA) on rat retinal neovascularization in retinae. Methods Retinal vein occlusion was induced by retinal photodynamic medthod in 25 Sprague-Dawley rats. Rac1-siRNA vector DNA was injected into the vitrous of one eye of those rats (gene intervention group), and empty vector DNA was injected into the fellow eye (blank control group). Rac1-siRNA vector was injected in other 25 SD rats without retinal vein occlusion (blank intervention group). Two weeks after injection, fluorescein isothiocyanate (FITC)-dextran was perfused into the hearts of all the rats, and the retinal wholemount was made to observe the neovascularization. The numbers of endothelial cells which break through the internal limiting membrane were counted after hematoxylin-eosin staining. Results A massive of neovascularization and FITC leakage were found in blank control group. Small part of neovascularization and a little FITC leakage were observed in the gene intervention group. Retinal vessels were normal in blank intervention group. Compared with blank contrast group and blank intervention group, the difference of the mean numbers of endothelial cells which broke through the internal limiting membrane in the gene intervention group was significant（t=? P=0.000??lt;0.05）. Conclusion Rac1-siRNA can inhibit retinal neovascularization induced by retinal vein occlusion in rats.