Studies showed that Toll like receptors (TLRs) play an important role in the development of retinal angiogenesis.However, the effect and mechanism of TLR4 affecting angiogenesis in oxygen induced retinopathy (OIR) had not been clarified yet.
This study aimed to investigate the mechanism of TLR4 in oxygen induced retinal neovascularization in mice.
Eighteen 7-day-old (P7) C57BL/6J mice were randomly divided into normoxic group, OIR model group and OIR+ lipopolysaccharide (LPS)-EB group.OIR models were established by exposing the mice with maternal mouse to 75% oxygen environment for 5 days, then 1 mg/kg LPS-EBO and PBS were intraperitoneally injected in P12 mice in the OIR+ LPS-EB group and OIR model group, respectively.Whole retinal flatmounts of P17 mice were prepared and Lectin staining was performed to calculate the ratio of avascular and neovascular area to retina area.The frozen sections of the posterior ocular segment were prepared, and the number of activated microglial cell was detected by immunofluorescence technique, and the expressions of CD11b and TLR4 in the microglial cells as well as the levels of vascular endothelial growth factor (VEGF), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) were assayed by fluorescence double labeling method.The use and care of the experimental animals complied with ARVO Statement.
The distribution and morphology of retinal vessels were normal in P17 mice of the normoxic group, and avascular and new blood vessels cluster were found in the mice of the OIR group and OIR+ LPS-EB group.The ratio of avascular area was (18.47±1.32)% and that of the new blood vessel area was (3.29±0.85)% in the OIR+ LPS-EB group, which were increased in comparison with (15.78±1.44)% and (1.77±0.19)% of the OIR group (t=3.36, P=0.01; t=4.22, P=0.00). Immunofluorescence results displayed a few Iba1+ microglia cells in the normoxic group, the cells number were significantly higher in the OIR+ LPS-EB group than that in the OIR group ([95.50±4.77]/5 mm versus [74.83±4.17]/5 mm; t=8.00, P<0.01). TLR4+ microglia cells were few in the normoxic group, and the cells number were evidently higher in the OIR+ LPS-EB group than that in the OIR group ([49.50±6.38]/5 mm versus [28.17±6.24]/5 mm, t=5.86, P<0.01). The number of microglia cells coexpressing CD11b with VEGF/IL-1β/TNF-α were (1.17±0.75) /5 mm, 0 and 0 in P17 mice of the normoxic group, respectively, and the cells number in the OIR+ LPS-EB group was more than that in the OIR group ([44.50±8.78]/5 mm versus [28.50±5.61]/5 mm, F=44.01, P<0.01; [24.10±6.49]/5 mm versus [16.00±3.46]/5 mm, F=11.31, P<0.01; [33.83±14.82]/5 mm versus [23.00±2.83]/5 mm, t=19.92, P<0.01).
TLR4 promotes retinal neovascularization probably by activating specific signaling pathways downstream of microglia cells and accelerating the release of vascular growth factors and inflammatory factors.