Objective To observe the efficacy of the anti-tumor necrosis factor-alpha; monoclonal antibody (TNF-alpha; MCAb) in the treatment of experimental autoimmune uveoretinitis (EAU). Methods EAU animal models were induced by interphotoreceptor retinoid-binding protein (IRBP) R16 peptide with immunization. The rats were divided into 2 groups according to the injection times. TNF-alpha; MCAb was administered intravenously on day 6 or 4, 6 and 8 post-immunization respectively, and then to observe the clinical expression by slit-lamp microscope. Meanwhile, take the rats which did not accept TNF-alpha; MCAb as control group. Delayed type hypersensitivity (DTH) responses were measured on day 13 post-immunization of IRBP R16; the rats were killed on day 14 post-immunization of IRBP R16, and then enucleated the eyes for histopathological examination. To detect the cytokine level of IFN-gamma;, IL-4 in serum and IFN-gamma; in aqueous humor by enzyme-liked immunosorbent assay (ELISA) on day 14 post-injection. The hyperplasia responses of antigen specific lymphocyte of draining lymph node cells were detected. Results The TNF-alpha; MCAb group had mitigated ocular inflammation and decreased pathological grades compared with the control group; the IFN-gamma; concentrations in aqueous humor and serum were decreased, IL-4 was increased in serum; DTH responses were decreased; the hyperplasia responses of draining lymphocytes to IRBP R16 peptide were decreased, all the differences were statistically significant (P＜0.01). The rats accepted TNF-alpha; MCAb thrice had much better curative effect than the rats injected once (P＜0.05). Conclusions Injection of TNF-alpha; MCAb can inhibit ocular inflammation and specific immune cells of EAU remarkably and change the Th1/Th2 balance. Many times injections of TNF-alpha; MCAb were more effective than once.
Objective To observe the regulation effect of transforming growth factor alpha (TGFalpha;) on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muuml;ller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7～10 day, and then cultured Muuml;ller cells according to literature. The 3～4 generation cultured cells of the same primary cell were divided into two groups at random: ① TGFalpha; group: maintained in different concentrations of TGFalpha; as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;② Control group: cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFalpha; on GLAST activity in Muuml;ller cells were observed by L-3H-glutamate uptake detection; the expression of GLAST mRNA in Muuml;ller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFalpha; concentration, both L3H glutamate uptake and GLAST mRNA expression were increased. The L-3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFalpha; on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant (Plt;0.05). Conclusion TGF-alpha; can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.
Objective To investigate the effect of doxycycline on the proliferation and vasculogenic mimicry in retinoblastoma (RB) cell line in vitro. Methods RB cell line were tested for their ability to form perfusable tubular networks in 3D culture with doxycycline in the concentrations ranging from 5 to 20 mg/L, and CoCl2 was used as chemical hypoxia-inducing reagent to mimic tumor hypoxic microenvironment. The effect of doxycycline on proliferation were detected by MTT assay in vitro, and the effect on tube formation of RB cells were detected by tube-like structure formation assay and PAS staining. The mRNA levels of MMP2 and MMP9 at different hypoxic culture and different doxycycline concentrations were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Results The micrograph showed that RB cells linked each other to form cavity and network tructure in 3D culture. the number of tubules in doxycycline group were significantly lower than which in the control group in the concentrations ranging from 5 to 20 mg/L (Plt;0.001).OD of doxycycline group was significantly lower than which in the control group (t=15.320，Plt;0.01) , The proliferation of RB cells had a negative correlation with the concentration of doxycycline (r =-0924, Plt;001). The levels of MMP2 and MMP9 mRNA of RB cells under hypoxia were significantly higher than which in the control group (t=16.469，Plt;0.01). As the concentration of doxycycline increased, the expression of MMP-2 and MMP-9 decreased. The result of double staining also showed that VM, formed by CD34negative and PASpositive tumor cells, existed in 12 simples of retinoblastoma. Conclusion RB cells have the capacity of selfmetamorphosing and vasculorizing in 3D culture. Doxycycline can inhibit their proliferation and vasculogenic mimicry formation in vitro by downregulating the expression of MMP-2 and MMP-9 .
Objective To investigate the dose-dependent relationship of bone marrow mesenchymal stem cells(MSCs) transplantation in improving ischemic myocardial dysfunction? in a rat ischemic heart model. Methods Myocardial infarction was induced in 32 inbred F344 rats by acute ligation of the left anterior descending(LAD) coronary artery. One week after ligation, the ratswere randomized? into four equal groups, with eight rats in each group. Equal volume Iscove’s modified Dulbecco’s medium was injected in the control group, 1×103(group 1), 1×105(group 2), and 1×107(group 3) 5-bromodeoxyuridine (BrdU) labeled bone marrow MSCs were injected into the infarcted myocardium. Cardiac function was evaluated by ultrasound before the ligation of the LAD, before the transplantation and the 4th week after transplantation. The expressions of BrdU,Connexin43,Myosin heavy chain β(MHC), and smooth muscle actin α(α-SMA) were detected by immunofluorescence and immunohistochemistry at the 4th week after transplantation. The amount of functional vessels stained by α-SMA was counted simultaneously. Results At the 4th week? after transplantation, the ejection fraction(EF) in goup 2 was more significantly improved than that in group1(0.54±0.20 vs. 0.34±0.16, P＝0.004) and EF in group 3 was more significantly improved than that in group 2(0.71±0.24 vs. 0.54±0.20,P＝0.018), whereas no significant difference between group 1 and control group was detected (0.34±0.16 vs. 0.36±0.15,Pgt;0.05). The BrdU labeled MSCs could be found in host myocardium. The number of cells in group 2 by double staining both for BrdU and for MHC observed in ischemic myocardium were significantly more than that in group 1? (323.20±91.62 n/HP vs. 51.75±27.58 n/HP,P＝0.049) and the same was true between group 3 and group 2(409.75±106.65 n/HP vs. 323.20±91.62 n/HP,Plt;0.001), whereas the result of control group was negative.The majority of transplanted cells were found positive staining both for MHC and for Connexin43 in all groups. There were lots of positive staining of α-SMA whose form were partly irregular in ischemic myocardium indicating that there was neovascularization in group1 and control group. More neovascularization in group2 was found than that in group 1 (28.38±12.79 n/HP vs. 22.75±9.07 n/HP, P＝0015) and more neovascularization in group 3 was found? than that in group 2 (35.63±13.27 n/HP vs. 28.38±12.79 n/HP, P＝0.002) . Conclusion Transplanted into infarcted myocardium, bone marrow MSCs may have significant and dose-dependent potential for cardiomyogenesis with functional recovery from myocardial ischemia.
Objective To observe the inhibition effect of selective cyclooxygenase2 inhibitor（celecoxib）on the experimental choroidal neovascularization（CNV）. Methods Thirty 8-10 weeks old healthy male Brown-Norway（BN）rats were randomly divided into the control, laser and celecoxib group,with 10 rats in each group. At the dosage of 50 mg/kg, celecoxib was gavaged twice per day. After 7 days, experimental CNV was induced by Krypon laser on laser group and celecoxib group. Fundus fluorescein angiography (FFA) was performed on days 3, 7,14,21,30 after laser photocoagulation.On days 21 after photocoagulation, 5 rats in each group were sacrificed and the relative thickness of CNV membranes, the expression of COX-2, vascular endothelial growth factor(VEGF) and matrix metalloproteinase-2(MMP-2) were studied by histopathologic or immunohistochemistry examination.Results On days 21 after photocoagulation, the incidence of CNV in the celecoxib group is significantly lower than that in the laser group (chi;2=7.1068,P=0.0077); the relative thickness of the CNV membranes in the celecoxib group is reduced 41.38% compared to the laser group, the difference is statistically significant (t=16.760 0，P=0.0000).COX-2,VEGF and MMP-2 expression in the CNV membrane of celecoxib group were significantly lower than in control group (t=5.710 0,5.840 0, 8.020 0; P=0.000 0); the COX-2, VEGF and MMP-2 expressions in choroid and retina of control group were weak. Conclusion Prophylactic celecoxib can reduce the expression of VEGF and MMP-2 by inhibiting COX-2, and prevent the CNV induced by laser photocoagulation.
Objective To observe the effects of intravenous thrombolysis with urokinase for central retinal artery occlusion (CRAO). Methods A total of 115 CRAO patients diagnosed by fluorescence fundus angiography (FFA) were enrolled in this study. The patients included 61 males and 54 females, with a mean age of (56.7plusmn;15.2) years (from 41 to 75 years). The duration ranged from 1 to 30 days. All the patients were affected unilaterally. All the patients were received the treatment of intravenous thrombolysis with urokinase (3000 U/kg, two times per day, continuous treatment for six to seven days) and retrobulbar injection of dexamethasone 2.5 mg (one time per day, continuous treatment for 14 days). Following that, 1.2 mg/kg brain protein hydrolysate (nerve nutrition) and 360 mg troxerutin (vasodilator) were given by intravenous drip (one time per day, continuous treatment for 14 days). Effectiveness of the thrombolytic and subsequent treatments including the recovery of vision and retinal arterial filling time before and after treatment were observed. Comparing the visual acuity of post-treatment and pre-treatment, improving three lines or more is considered as effective markedly, improving two lines as effective, no change or a decline as no effect. With FFA as the retinal circulation recovery index, the arm-retinal circulation time (A-Rct ) le; 15s and all branches of central retinal artery were filled with fluorescence within 2s filling (normal) as effective markedly; A-Rct improved but was in 15 - 20s range, all branches of central retinal artery were filled with fluorescence within 3～8s as effective; A-Rct improved but was still ge; 21s, all branches of central retinal artery were filled with fluorescence within ge;9s as no effect. The relationship between age, gender, the disease course, subsequent treat time and curative effectiveness were analyzed. Results There were 79 patients were examined for FFA again after thrombolysis treatment which including 11 patients with complete obstruction and 68 patients with incomplete obstruction. In 11 patients with complete obstruction, eight patients showed that optic disc vascular retrograde filling disappeared, A-Rct was 28-54s, and the filling time from retinal artery to tip was 18 - 55s; three patients showed persistent optic disc vascular retrograde filling within 3 - 4 minutes of FFA. In 68 patients with incomplete obstruction, A-Rct returned to normal in 35 patients (51.4%), effective in 18 patients (26.5%) and no effect in 15 patients (22.1%). Retinal circulation time was shorter than that before thrombolysis treatment (chi;2=11.4, Plt;0.05). Comparison of distribution of visual acuity before and after thrombolysis treatment, the difference was statistically significant (chi;2=12.1, Plt;0.05). Comparison of distribution of final visual acuity after subsequent treatment with that of after thrombolysis treatment, 48 eyes improved two lines or more, the efficiency was 41.7%, the difference was statistically significant (chi;2=14.6, Plt;0.05). Comparison to that of before treatment, vision changes showed effect markedly in 58 patients (50.4%), effective in 35 patients (30.4%), no effect in 22 patients (19.2%), the difference was statistically significant (chi;2=44.5, Plt;0.05). Comparison the average age to that of effective, valid and invalid patients, the difference was not statistically significant (t=0.98, 1.17, 0.55; Pgt;0.05). There was no relationship between effectiveness and gender (chi;2=2.6, Pgt;0.05). In 76 patients with duration within seven days, 43 patients were effective markedly and 22 patients were effective, the efficiency was 85.5%. In 25 patients with duration of 8 - 15 days, 11 patients were effective markedly and eight patients were effective, the efficiency was 76.0%. In 34 patients who received subsequent treatment 8 - 14 days, 18 patients were effective markedly and nine patients were effective, the efficiency was 79.4%. In 51 patients who received subsequent treatment 15-21 days, 27 patients were effective markedly and 18 patients were effective, the efficiency was 88.2%. Conclusion Intravenous thrombolysis with urokinase was effective in the treatment of CRAO.
Objective To investigate the retinal toxicity and verify the safe dose of intravitreal injecting fluconazole. Methods Twelve healthy adult white rabbits were divided at random into 6 groups：a normal control group and 5 groups received intravitreal injection of a single dose of fluconazole ranging from 10 to 200 mu;g respectively.Retinal toxicity was examined by ophthalmoscopy, electroretinography, light and transmission electron microscopy (TEM) on the third and fourteenth day after injection. Results The ultrastructures of the retinal tissues of the normal control group and fluconazole 10～150 mu;g groups were normal on the third and fourteen day after injection.The light microscopy and TEM showed that cells of all the retinal layers in the 200 mu;g group revealed apparent degenerative changes on the fourteenth day after injection, and the light microscopic picture showed the vacuolar degeneration of outer segments of photoreceptors, the nuclei in outer nuclear layer drop out into inner segments, the vacuolar degeneration of nerve fiber layer, and the proliferation of pigment epithelium. TEM revealed expansion of paranucl eus space and karyopyknosis of the bipolar cells, the swelling of nerve fibers and disappearance of the synapses in the inner plexiform layer, the vacuolation and disappearance of microvilli of the pigment epithelium cells. Conclusion The safe dose of fluconazole injected intravitreally should be 100～150 mu;g. (Chin J Ocul Fundus Dis，2000，16：139-212)
ObjectiveTo evaluate the effect of intravitreous injection with triamcinolone acetonide (TA) on macular edema.MothodHaving been examined by ophthalmoscopy, optic coherent tomography (OCT), retinal thickness analyzer (RTA), and fundus fluorescein angiography (FFA), 33 patients (37 eyes) with diffused and (or) cystoid macular edema caused by diabetes and retinal venous occlusion were intravitreously injected with 0.1 ml triamcinolone acetonide (40 mg/ml). During 1-9 month followup period, the visual acuity, intraocular pressure, inflammatory extent, manifestation of lens and fundus were observed, the retinal thickness was examined by OCT and RTA, and vascular leakage were detected by FFA.ResultsMacular thickness was (244.07±118.80), (195.53±57.70), and (181.42±54.79) μm respectively 1, 2, 3 months after treatment; while macular thickness was (724.35±227.41) μm before the treatment. The difference was statistically significant （t =10.72, 12.84, 13.90; P lt;0.001）. The visual acuity was 0.39±0.19, 0.45±0.24, and 0.43±0.21 respectively, comparing with the visual acuity before the treatment (0.20±0.16), the difference was statistically significant （t =4.445, 4.349, 3.474; P lt;0.001, lt;0.001, 0.03）；The result of FFA showed less leakage of fluorescein and proliferative lesion. Four pateints had the ocular pressure ≥25 mm Hg (1 mm Hg=0.133 kPa) in 9 who had ≥20 mm Hg. Recurrence of macular edema was found in 4 eyes of 3 patients 4 and 6 months after the treatment, respectively. No infection or aggravation of lenticular turbidness occurred.ConclusionIntravitreous injection with TA can be used to treat macular edema due to diabetes and retinal venous occlusion, and recurrence of macular edema or increase of intraocular pressure may occur in some patients.(Chin J Ocul Fundus Dis, 2005,21:205-208)
What drug dosage range is appropriate for treatment? What drug dosage range can maximally reduce the incidence of adverse drug reaction (ADR)? The gold zone method as a new method of evidence-based medical research was proposed to study those two blind areas of drug dosage in this article. Studying the dose-effect relationship, taking gold zone as the middle range and dividing empirical range into 3 sections were the key to study design. The evidence-based survey with extremely large sample showed a U-shaped rule existing between the antibiotics’ dosage and the incidence of ADR; and the dosage in gold zone appeared at the bottom of U-shaped curve. The gold zone method for determining dosage is a special breakthrough currently for solving those two blind areas of drug dosage
Objective To investigate the retinal toxicity and verify the safe dose of intravitreal injection of nonsteroid anti-inflamatory drug,diclofenac sodium.Methods Twenty-eight healthy adult white rabbits were divided at random into 7 groups and received in every right eye the intravitreal injection of a single dose of diclofenac sodium solution ranging from 0.4-0.1 mg/0.1ml respectively ,the left eyes were regarded as conreol ones.Before injection and on the 1st,3rd,7th,14th,21st,and 28th day after injection the electroretinography on both eyes was examined.On the 28th day after injection the retinas of two rabbits of every group were examined by using light microscopy.On the 10th and 30th day after injection the retinal tissues around the optic nerve sisk of two eyes from every group at random were tested by using transmission electron microscopy.Results The retio of amplitude ofb wave of electroretinography in 0.4mg and 0.5mg groups had no sighnificant difference from groups before injection,the retinal tissues showed no structural changes in light and ecectron microscopy examination.The ratio of amplitude ofb wave of photoptic electroretinogrphy in 0.6mg groups in the early stage after injection was markedly reduced（P＜0.05）and returned to that before injection with time,reversible change of the edematou retina was discovered.The ratio of amplitude of b wave of electroretinography in 0.7-1.0mg groups was distinctly descreaded after injection（P＜0.05 or P＜0.01）,the cells of all the retinal layer revealed apparent and irreversible damage.Conclusion The largest dose of safety of intravitreal diclofenac sodium should be not more than 0.6mg.The toxic effect of intravitreal diclofenac sodium on retina is concerned mostly to cones and rods.