From the results of this experiment, it showed that the implanted tendon was gradually extruded from the tibia hole and attached to the periosteum. The dominant breeding of tissue cells, cytodynamics, the perimeter ratio of tendon/bone and the effect of revascularization were discussed in detail.
Objective
Methods
Ninety male Wister rats were randomly divided into normal control group, diabetic group and FTY720 group, thirty rats in each group. Diabetes was induced by giving a single intraperitoneal injection of streptozocin. FTY720 group was administered with FTY720 at a dose of 0.3 mg/kg by oral gavage daily for 3 months after establishment of diabetes. All rats were used for experiments following intervention for 3 months in FTY720 group. Immunohistochemical staining was used to observe the expression and distribution of intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1), and the positive cells were counted. Real-time reverse transcription PCR was used to measure mRNA expression of ICAM-1 and VCAM-1. Fluorescein isothiocyanate-Concanavalin A perfusion was used to detect retinal leukocytes adhesion. Evans blue (EB) perfusion was used to analyze retinal vascular permeability. Immunofluorescence staining was used to detect retinal inflammatory cells infiltration.
Results
In diabetic group, both ICAM-1(t=12.81) and VCAM-1 (t=11.75) positive cells as well as their mRNA expression (t=16.14, 9.59) were increased compared with normal control group, with statistical significance (P < 0.05). In FTY720 group, both ICAM-1(t=-9.93) and VCAM-1 (t=-6.61) positive cells as well as their mRNA expression (t=-15.28, -6.10) were decreased compared with diabetic group, with statistical significance (P < 0.05). Retinal leukocytes adhesion (t=16.32) and EB permeability (t=17.83) were increased in diabetic group compared with normal control group, while they were decreased in FTY720 group compared with diabetic group(t=-9.93, -11.82),with statistical significance (P < 0.05). There were many CD45 positive leukocytes infiltration in retina of diabetic group, including CD11b positive macrophage/activated microglia, while both of them were little in FTY720 group.
Conclusions
FTY720 can decrease retinal leukocytes adhesion, reduce retinal vascular permeability and inflammatory cells infiltration, which is associated with down-regulation of ICAM-1 and VCAM-1.
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.
ObjectiveTo investigate the effects of transforming growth factor-β (TGF-β) in choroidal neovascularization (CNV) induced by laser in mice.
Methods Eighty male C57BL/6J mice at the age of 6-8 weeks old were randomly divided into the normal control, photocoagulation model, photocoagulation with phosphate buffered saline (PBS control group) and photocoagulation with TGF-β receptor inhibitor groups (TGF-β receptor inhibitor group), twenty mice of each group. Fundus argon laser photocoagulation was performed in the photocoagulation model group, PBS control group and TGF-β receptor inhibitor group to induce CNV. One week, two, three and four weeks after the laser procedure, fundus fluorescein angiography (FFA) was carried out in the normal control or photocoagulation model groups to observe CNV formation dynamically. Western blot was used to analyze the expressions of TGF-β in the retina from the mice of normal control or photocoagulation model groups, and VEGF or TNF-α in the retina of normal control, PBS control or TGF-β receptor inhibitor groups. The CNV areas of each group were evaluated by using fluorescein stain on retinal pigment epithelium (RPE)/choroid flat mounts after two weeks of photocoagulation.
ResultsThe FFA results showed the retinal vessels centered on the optic disc and arranged radially, while the choroidal vascular present network distribution in the normal control mice. Significant leakage of fluorescein showed discoid strong fluorophore in photocoagulation sites of retina at one week after photocoagulation. The quantitative analysis results of Western blot demonstrated that the TGF-β protein expression levels in retina of photocoagulation model mice gradually increased with time passing. The protein expression levels of TGF-β were significant differences in the photocoagulation model group comparing with the normal control group (F=13.042, P < 0.05). The protein expression levels of TNF-α (F=14.721, 17.509) and VEGF (F=18.890, 11.251) increased significantly in retina of PBS control or TGF-β receptor inhibitor groups when compared with that of normal control group at one week, two, three and four weeks after photocoagulation, and the differences were both statistically significant (P < 0.05). Compared with PBS control group, the protein levels of TNF-α and VEGF in retina from TGF-β receptor inhibitor group were significantly reduced, the differences was statistically significant (F=21.321, 16.160, P < 0.05). Two weeks after laser photocoagulation, a distinct reduction in CNV lesion size in the TGF-β receptor inhibitor group mice when compared to PBS or normal control groups, the differences was statically significant (F=4.482, P < 0.05).
ConclusionTGF-β may promote CNV formation by up-regulating both TNF-α and VEGF protein expressions, the application of its specific inhibitor is able to reduce CNV progression.
Objective
To systematically review the effectiveness and model building process of heparin treatment for animal model with smoke inhalation injury.
Methods
Databases including PubMed, EMbase, CBM, CNKI, VIP and WanFang Data were searched to collect animal experiments about the treatment of heparin for animal model with smoke inhalation injury from inception to November 2016. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies. Then meta-analysis was conducted by RevMan 5.3 software.
Results
A total of nine studies involving 11 animal experiments were included. The results showed that building animal model with smoke inhalation injury were through burning of cotton towels or pine sawdust by sheep or rats below 40℃. The results of meta-analysis showed that there was no significant difference in mortality rate between two groups (heparin group vs. control group: RR=0.38, 95%CI 0.14 to 1.05, P=0.06; heparin plus DMSO group vs. DMSO group: RR=0.10, 95%CI 0.01 to 1.51, P=0.10). In addition, the pulmonary artery pressure (MD=–3.31, 95%CI –4.51 to –2.11, P<0.000 01), wet to dry weight ratio (MD=–0.90, 95%CI –1.19 to –0.61, P<0.000 01), and lung water content (MD=–1.18, 95%CI –1.67 to –0.70, P<0.000 01) of the experimental group were lower than those in the control group. PaO2/FiO2 after 12 hours (MD=131.00, 95%CI 59.54 to 202.46, P=0.000 3), PaO2/FiO2 after 24 hours (MD=114.00, 95%CI 60.56 to 167.44, P<0.000 1), PaO2/FiO2 after 48 hours (MD=46.00, 95%CI 20.62 to 71.38, P=0.000 4) were higher than those in the control group. However, there was no significant difference in coagulation function between both groups.
Conclusion
The current evidence shows that the establishment of animal model of smoke inhalation injury is still lack of standard method. Heparin can decrease pulmonary artery pressure and lung water content in animal models with smoke inhalation injury. Due to the limited quality and quantity of included studies, the above conclusions are still needed to be verified by more high quality studies.
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.
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.
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.
Objective
To investigate the protective effect of Niacin on blood-retina barrier (BRB) in diabetic rats and related mechanism.
Methods
The male Wistar rats (60) were divided into control (CON) group, diabetes (DM) group and Niacin-treated (NA) group, 20 rats in each group. Rats diabetes models were induced with streptozotocin injection. Niacin (40 mg/kg·d) was administrated orally everyday in Niacin-treated group until sacrificed after 3 months. Pathological outcomes, total cholesterol (TC) and high-density lipoprotein (HDL) were evaluated at month 3. Optical microscopy was used to observe the retinal structure. The integrity of BRB and the vascular permeability was quantified by analyzing albumin leakage using Evans blue (EB) method. The relative expressions of Claudin-5, Occludin, zonula occluden (ZO)-1 and GPR109A mRNA in rat retinas were detected by reverse transcription PCR (RT-PCR) and relative expression of GPR109A, tumor necrosis factor (TNF)-α and interleukin (IL)-6 by Western blot.
Results
Compared to CON group, the TC content was increased and HDL content was decreased in DM group (t=4.034, 5.831; P < 0.05). Compared to DM group, the TC content was decreased and HDL content was increased in NA group (t=6.868, 3.369; P < 0.05). The retinal structure of CON group was normal. Pathological changes were found in the DM group, such as tumescent nuclei and disorganized structures. The retinal structure of NA group was similar to the control group. Evans blue dye that the microvascular leakage in DM group was increased compared with CON group (t=24.712, P < 0.05), while in NA group was decreased compared with DM group (t=16.414, P < 0.05). The mRNA expression of Occludin, Claudin-5, ZO-1 in DM group were decreased compared with CON group (t=11.422, 12.638, 12.060; P < 0.05), while in NA group were increased compared with DM group (t=5.278, 3.952, 8.030; P < 0.05). The mRNA expression of GPR109A in NA group were increased compared with DM group (t=5.053, P < 0.05). The protein expression of GPR109A, IL-6, TNF-αin DM group were increased compared with CON group (t=4.915, 11.106, 6.582; P < 0.05). Compared to DM group, the protein expression of GPR109A was increased (t=5.806, P < 0.05), while the protein expression of IL-6 and TNF-α were decreased (t=10.131, 5.017; P < 0.05).
Conclusion
Niacin has the protective effect for BRB by up-regulating GPR109A expression which may suppress inflammation.
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.
