X. Liang

TMP prevents retinal neovascularization and imparts neuroprotection in an oxygen-induced retinopathy model.

PURPOSEnTo evaluate the effects of tetramethylpyrazine (TMP) on retinal neovascularization (NV) and neuroprotection in an oxygen-induced retinopathy (OIR) model.nnnMETHODSnNeonatal C57BL/6J mice were subjected to 75% oxygen from postnatal day 7 (P7) to P12 and then returned to room air. TMP (200 mg/kg) or normal saline was given daily from P12 to P17. Immunostaining, HE staining, TUNEL assay, and RT-PCR were used to assess the effects of TMP on retinal neurovascular repair.nnnRESULTSnTMP effectively prevented pathologic NV and accelerated physiologic revascularization by enhancing the formation of endothelial tip cells at the edges of the repairing capillary networks and preserving the astrocytic template in the avascular retina. TMP also prevented morphologic changes and significantly decreased TUNEL-positive cells in the avascular retina by rescuing neurons such as amacrine, rod bipolar, horizontal, and Müller cells. In TMP-treated mice retinas, there was a less obvious loss of amacrine cell bodies and their distinct bands; the number of both rod bipolar and horizontal cell bodies, as well as the density of their dendrites in the outer plexiform layer, was greater than that in OIR control mice. TMP not only decreased the loss of alignment of Müller cell bodies and distortion of processes but reduced the reactive expression of GFAP in Müller cells. Furthermore, HIF-1α and VEGF mRNA expression were downregulated in TMP-treated mice retinas.nnnCONCLUSIONSnTMP improved neurovascular recovery by preventing NV and protecting retinal astroglia cells and neurons from ischemia-induced cell death partially due to its downregulation of HIF-1α and VEGF mRNA expression.

Madecassic Acid protects against hypoxia-induced oxidative stress in retinal microvascular endothelial cells via ROS-mediated endoplasmic reticulum stress.

Madecassic acid (MA) is an abundant triterpenoid in Centella asiatica (L.) Urban. (Apiaceae) that has been used as a wound-healing, anti-inflammatory and anti-cancer agent. Up to now, the effects of MA against oxidative stress remain unclear. In this study, we investigated the effect of MA and its mechanisms on hypoxia-induced human Retinal Microvascular Endothelial Cells (hRMECs). hRMECs were pre-treated with different concentrations of MA (0-50μM) for 30min before being incubated under hypoxia condition (37°C, 5% CO2 and 95% N2). Cell apoptosis was evaluated with MTT assay and TUNEL staining, and the expression of apoptosis- and endoplasmic reticulum (ER) stress-related molecules was assessed with western blotting and RT-PCR analysis. Intracellular ROS level was evaluated using DCFH-DA. Intracellular malondialdehyde (MDA), dehydrogenase (LDH), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD) were evaluated using related Kits. Activating transcription factor 4 (ATF4) nuclear translocation was assessed with western blotting analysis and immunofluorescence staining. MA significantly reduced oxidative stress in hypoxia-induced hRMECs, as shown by increased cell viability, SOD and GSH-PX leakage, decreased TUNEL- and ROS-positive cell ratio, LDH and MDA leakage, caspase-3 and -9 activity, and Bax/Bcl-2 ratio. In addition, MA also attenuated hypoxia-induced ER stress in hRMECs, as shown by reduced mRNA levels of glucose-regulated protein 78 (GRP78), C/EBP homologous transcription factor (CHOP), protein levels of cleaved activating transcription factor 6 (ATF6) and inositol-requiring kinase/endonuclease 1 alpha (IRE1α), phosphorylation of pancreatic ER stress kinase (PERK) and eukaryotic initiation factor 2 alpha (eIF2α), cleaved caspase-12 and ATF4 translocation to nucleus. The current study indicated that the regulation of oxidative stress and ER stress by MA would be a promising therapy to reverse the process and development of hypoxia-induced hRMECs dysfunction.

Increased sCD200 Levels in Vitreous of Patients With Proliferative Diabetic Retinopathy and Its Correlation With VEGF and Proinflammatory Cytokines

PURPOSEnThe purpose of this study was to determine the levels of sCD200 expression in the vitreous of proliferative diabetic retinopathy (PDR) patients and to clarify its correlation with different vitreoretinal conditions, VEGF and its receptors, and proinflammatory cytokines.nnnMETHODSnThe expression of sCD200, VEGF and its receptors, and other proinflammatory cytokines were examined by using ELISA. Clinical stratification was performed on patients with different vitreoretinal conditions for correlation analysis.nnnRESULTSnThe vitreous levels of sCD200 were significantly higher in the PDR group (182.2 ± 17.63 pg/mL) compared with those in the control group (56.86 ± 6.573 pg/mL; P < 0.0001). The venous blood levels of sCD200 were 26.71 ± 4.32 pg/mL in the PDR group and 19.94 ± 3.87 pg/mL in the control group (P = 0.2614). The vitreous levels of sCD200 were significantly elevated in PDR patients with diabetic macular edema (DME; 266.9 ± 28.82 pg/mL) or traction retinal detachment (TRD; 256.9 ± 34.50 pg/mL) compared with the PDR group without DME (136.9 ± 15.13 pg/mL; P < 0.0001) or TRD (146.9 ± 15.97 pg/mL; P = 0.0024). The vitreous levels of CCL2, CXCL4, CXCL9, CXCL10, VEGF, sVEGFR-1, sVEGFR-2, IL-6, IL-8, IL-10, and IL-18 were also elevated significantly in the PDR group. Statistical association was found between sCD200 levels and VEGF (r = 0.6566, P < 0.0001), sVEGFR-1 (r = 0.5574, P = 0.006), sVEGFR-2 (r = 0.3605, P = 0.0362), CCL-2 (r = 0.6001, P = 0.0002), IL-6 (r = 0.5704, P = 0.0004), IL-8 (r = 0.3712, P = 0.0307), and IL-10 (r = 0.3618, P = 0.0355).nnnCONCLUSIONSnExpression of sCD200 may contribute to retinal angiogenesis by interacting with VEGF-mediated inflammatory response and represents a potential therapeutic target for the patients with PDR.

Anti-angiogenic and anti-inflammatory effect of Magnolol in the oxygen-induced retinopathy model

ObjectiveIn the present study, we investigated the effects of Magnolol on the retinal neovascularization (RNV) and local glial cells in an oxygen-induced retinopathy (OIR) model and explored their molecular mechanisms.Materials and methodsNeonatal C57BL/6J mice were subjected to 75xa0% O2xa0±xa05xa0% from postnatal day (P) 7 to P12 and subsequently returned to room air. Mice were injected with 25xa0mg/kg Magnolol intraperitoneally once a day from P12 to P17, then retinas were harvested and flat-mounted to assess the retinal vessels, astrocytes and microglia. To clarify the molecular mechanisms of Magnolol, we observed the level of inflammatory cytokines such as interleukin (IL)-1β, IL-6, monocyte chemoattractant protein-1, tumor necrosis factor-α, and analyzed the hypoxia-inducible factor (HIF)-1α/vascular endothelial growth factor (VEGF) pathway in OIR mice.ResultsIntraperitoneal administration of Magnolol resulted in significant reduction of RNV without retinal toxicity or perturbation of developmental retinal angiogenesis. In addition, Magnolol preserved the astrocyte morphology and diminished the activation of microglia. Moreover, Magnolol down regulated the expression of inflammatory cytokines and inactivated the HIF-1α/VEGF pathway.ConclusionsThese results indicated that Magnolol might have potential for the treatment of pathological retinal angiogenesis and glial dysfunctions via anti-inflammation and inhibition of HIF-1α/VEGF pathway.

Diacylglycerol Kinase (DGK) Inhibitor II (R59949) Could Suppress Retinal Neovascularization and Protect Retinal Astrocytes in an Oxygen-Induced Retinopathy Model

Hypoxia-inducible factors (HIF) play a fundamental role in retinal neovascularization (NV) induced by low oxygen tension. In the presence of oxygen, the HIF-α subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases (PHD), which triggers HIF-α for degradation. In our present study, we examined the effect of R59949, the diacylglycerol kinase (DGK) inhibitor II, on the retinal NV and its potential mechanism in an oxygen-induced retinopathy (OIR) model. OIR model was induced by exposure of hyperoxia (75xa0% oxygen) to C57BL/6J mice from postnatal day 7 (P7) to P12 and then returned to room air. By intraperitoneal injection once a day (10xa0μg/g/day) from P12 to P17, R59949 not only effectively prevented pathologic NV but also preserved the astrocyte morphology. Furthermore, the expression of PHD-2 was upregulated and HIF-1α and vascular endothelial growth factor (VEGF) were downregulated in the retina of OIR mice following R59949 treatment. These findings suggested a potential possibility that R59949 suppressed retinal neovascular pathophysiology via PHD2/HIF-1α/VEGF pathway.

A comparing study of quantitative staining techniques for retinal neovascularization in a mouse model of oxygen-induced retinopathy

AIMnTo explore an efficient, practical and objective quantitative method to evaluate the retinal neovascularization in mouse model of oxygen induced retinopathy (OIR).nnnMETHODSnThirty C57BL/6J mice were explored in OIR model procedure. Eyes were removed for different staining methods including: (1) HE staining; (2) immunohistochemistry with Griffonia Simplicifolia Lectin(GSL); (3) Immunofluorescence with FITC labeled CD31 antibody; (4) Two-step immunofluorescence with purified-CD31 antibody; (5) FITC-Dextran perfusion combined with two-step purified-CD31immunofluorescence. Images of the retinal vasculature were analyzed by imaging software.nnnRESULTSnGSL immunohistochemistry could clearly demonstrate the deep and superficial capillary beds. FITC labeled CD31 Immunofluorescence was blurring with high fluorescence background which was hard to distinguish retinal neovascularization in some area. Excellent detail of neovascularization and preexistent retinal vessels was provided in two-step Purified-CD31 immunofluorescence group.nnnCONCLUSIONnGSL immunohistochemistry can clearly demonstrate neovascularization tufts in deep and superficial capillary beds. Immunofluorescence of specific antigen CD31 on vascular endothelium can selectively label the neovascularization of mouse retina. When combined with computer analysis software, it is an effective and objective quantitative method to evaluate the retinal neovascularization in OIR mouse model.

Endometriosis has no negative impact on outcomes of in vitro fertilisation in women with poor ovarian response

To compare the in vitro fertilisation (IVF) outcomes of poor ovarian responders among women with laparoscopically diagnosed minimal–mild endometriosis (Group A), moderate–severe endometriosis (Group B) and those without endometriosis (Group C). The comparisons were made separately for age groups younger than 35 years and 35 years or older.