Objective To observe the inhibitory effect of tetramethylpyrazine (TMP) on apoptosis of retinal neurons in oxygeninduced retinopathy (OIR). Methods 48 C57BL/6 mice were randomly divided into normal control group (n=18), OIR control group (n=18) and OIR TMP group (n=12). The mice of normal control group were raised in room air. From the postnatal day 7 (P7), mice of the other two groups were exposed to (75 3) % oxygen for 5 days and then returned to room air to establish OIR model. The mice of OIR TMP group received intraperitoneal injection of TMP (200 mg/kg) once a day from P12 to P16, meanwhile the mice of normal control group and OIR control group were injected with the same volume of normal saline containing of 0.1% DMSO. At P12, P14 and P17, the morphologic changes in retinal avascular zone and the number of retinal apoptotic cell were observed by HE staining and TUNEL assay. Results At P12, there were a few of chromatin condensation and pycnic nuclei in the inner nuclear layer (INL) of OIR control group. At P14, a great quantity (OIR control group) or some (OID TMP treated group) chromatin condensation and pycnic nuclei in the central INL were observed. At P17, the thickness of INL, inner plexiform layer (IPL) and outer plexiform layer (OPL) in the OIR control group were reduced; the thickness of INL, IPL and OPL in the OIR TMP group weas thinner than those in the normal control group and thicker than those in the OIR control group. At P12, the TUNEL-positive cells in the OIR control group was 6 times of the normal control group (F=587.217，P＜0.001). At P14, the difference of TUNEL-positive cells in three groups was significant (F=587.217，P＜0.001); the TUNEL-positive cells in the OIR control group was 28 times of the normal control group (t=49.813，P＜0.001); the TUNEL-positive cells in the OIR TMP group has reduced 50% compared with the OIR control group (t=42.434，P＜0.001). At P17, there was no significant difference in TUNEL-positive cells among the three groups (F=587.217，P＞0.05). Conclusions TMP can inhibit apoptosis of retinal cells in OIR significantly.
Objective To observe the influence of prolyl hydroxylase 2 (PHD2) expression on endothelial barrier dysfunction induced by high glucose in human retinal vascular endothelial cells (HRECs). Methods The HRECs were treated by different culture medium with various glucose concentrations (5 mmol/L glucose, 5 mmol/L glucose +25 mmol/L mannitol, 30 mmol/L glucose) as normal control group, mannitol control group and high glucose group, respectively. After the cells cultured for 24 and 48 hours, the protein levels of PHD2, hypoxia-inducible factor-1alpha; (HIF-1alpha;) and occludin was detected by Western blot; the expression of vascular endothelial growth factor (VEGF) in the supernatant was determined by enzymelinked immuno sorbent assay (ELISA); the transcription levels of PHD2, HIF-1alpha;, VEGF and occludin were determined by the reversetranscription polymerase chain reaction (RT-PCR); the paracellular permeability between endotheliums was detected by 7times;104 molecular weight FITCdextran. Results Compared with normal control group, the protein level of PHD2 in mannitol control group and high glucose group firstly decreased and then increased, the protein level of HIF-1alpha; increased while that of occludin decreased; the secretion of VEGF increased in high glucose group but not in mannitol control group (PHD2：F=7.618, 8.627;P＜0.05. HIF-1alpha;：chi;2=7.692, 7.652;P＜0.05. occludin：F=23.23, 7.317;P＜0.05. VEGF：F=10.768, 4.562; P＜0.05). Compared with normal control group, the mRNA levels of PHD2, HIF-1alpha;, VEGF and occludin in mannitol control group and high glucose group increased (PHD2：F=5.69, 14.27;P＜0.05. HIF-1alpha;：F=6.07, 10.47;P＜0.05. VEGF：F=12.31, 9.14;P＜0.05. occludin：F=8.77, 8.00;P＜0.05). Compared with normal control group, the paracellular permeability of mannitol control group and high glucose group increased (chi;2=20.57,F=56.09;P＜0.05). Conclusions High glucose induced altered expression of PHD2 which might play an important role in endothelial barrier dysfunction. The mechanism might be associated with HIF-1alpha; and VEGF.
Objective To investigate the expression and prolyl hydroxylase (PHD)2 in retina of diabetic rats．Methods Wistar rats were randomly divided into the control group (n=48) and the diabetes group (n=60). The rats in diabetes group were induced with streptozotocin (STZ) injection creating a diabetic retinopathy model. The same volume of citric acid buffer was injected into the rats in the control group. Fluorescence microscope was used to observe the retinal vasculature at one, three and six months after injection. Evans blue perfusion was used to detect the bloodretinal barrier (BRB) permeability. Immunohistochemical staining was used to observe the distribution of PHD-2 positive staining. Western blot was used to measure the protein expression of PHD-2, hypoxia inducible factor（HIF）-1alpha; and vascular endothelial growth factor (VEGF) every month from one to six months after injection. Results The vascularization was normal and form was clear in retina of rats in control group. The retinal blood vessels of rats in diabetes group showed significantly increased fluorescence. Compared with the control group, the BRB permeability was significantly increased in diabetes group (P＜0.05). Abundant expression of PHD-2 protein was detected in the inner layers of retina in control and diabetes group．Compared with the control group, the PHD-2 expression was decreased in diabetes group at one and two months after injection (t=16.230, 16.390;P＜0.05). The HIF-1alpha; expression was significantly increased in diabetes group at one, two and three months after injection (t=27.073, 36.709, 10.176; P＜0.05). The VEGF expression was significantly increased in diabetes group every month from one to six months after injection (t=13.547, 31.984, 21.897, 8.912, 9.019, 14.046; P＜0.05). Conclusions There is abundant expression of PHD-2 in the inner layer of retina in diabetic rats. PHD-2 may play an important role in diabetic retinopathy, which is correlated with VEGF．
Objective To observe the effect of intravitreal bevacizumab (IVB) for exudative age-related macular degeneration (eAMD), and analyze the reasons for treatment failure. Methods Eighteen eyes of 17 patients with eAMD who have been diagnosed by fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA) and optical coherence tomography (OCT) were enrolled in this openlabel, single treatment group and prospective study. The patients received the first IVB treatment after diagnosis, and received the 2nd, 3rd, 4th, 5th and 6th IVB treatment at 6, 12, 24, 36, 48 weeks after the first injection. The examinations of best corrected visual acuity (BCVA), intraocular pressure, slit lamp microscope, fundus photography, FFA, ICGA and OCT were performed before and after treatment. Non-responders were defined as patients who had BCVA loss more than 5 letters at week 52 compared with BCVA before treatment. The therapeutic effects of IVB for eAMD were observed. The central macular thickness (CMT), maximum retinal thickness (MRT) and morphological changes of FFA, ICGA and OCT before and after treatment were comparative analyzed. Results Of the 18 eyes, 12 eyes (66.67%) were effective to IVB, 6 eyes (33.33%) were non-responders. The average CMT and MRT of non-responders were 506.83, 635.33 mu;m before treatment, which decreased to 446.17, 563.67 mu;m at 52 weeks. They were reduced by 60.67 and 71.67 mu;m respectively, but there was no statistically significant differences (t=-1.572,-0.943; P=0.116, 0.345). FFA and ICGA examination found that the CNV lesions of 3 non-responder eyes located in the foveal region, and became scars after the 6th treatment. The other 3 non-responder eyes developed CNV repeatedly during the treatments, and after the 6th injections of bevacizumab the lesions were under control with less fluorescein leakage and macular retinal edema. ConclusionIVB treatment is effective for some eAMD patients, but is ineffective for patients with extensive expanded CNV and foveal CNV.