Overexpression and knockdown of MFG-E8 in microglial cells after transfection with a lentivirus.

<p>Four different transfections were used: M+, MFG-E8 overexpression using pGC-FU-MFG-E8; M-, MFG-E8 negative control for overexpression using the pGC-FU vector; R+, Knockdown expression using pFU-GW-RNAi-MFG-E8; R- negative control for knockdown expression using the pFU-GW Vector. (A) The transfection rate was over 80% in all experiments. (B) qRT-PCR analysis indicated that pGC-FU-MFG-E8 significantly enhanced microglial MFG-E8 expression, and pFU-GW-RNAi-MFG-E8 effectively knocked it down. The negative controls had no effect. (C) Western blot analysis of the cell lysate from the transfected microglial cells confirmed that MFG-E8 expression (about 47 kDa) was significantly enhanced or knocked down. Scale bar: 50 µm. ** <i>P</i><0.01.</p

The phagocytosis of PS-hRBCs and mRNA expression of TNF-α and IL-1β by microglial cell co-cultures.

<p>(A) Representative confocal images showing the phagocytosis of of prelabeled PSox-hRBCs (red). (B–B′) The internalization of the PSox-hRBCs (red) material into the microglial cell (labeled with GFP) can be seen clearly in the confocal z-stacked images. (C) Control microglial cells without transfection; M+, MFG-E8 overexpression of pGC-FU-MFG-E8; M-, pGC-FU transfection; R+, blocked MFG-E8 expression- pFU-GW-MFG-E8; R-, nonsense sequence in the pFU-GW. (D) Overexpression of MFG-E8 in microglial cells increased the phagocytosis of PS-hRBCs; phagocytosis rate dramatically decreased in knocked downs. (E–F) The mRNA expression of TNF-α and IL-1β increased in the MFG-E8 up-regulated microglial cell, but significantly decreased when MFG-E8 was knocked down. Scale bars in A–B: 20 µm; C–R-: 50 µm; ** <i>P</i><0.01.</p

The detection of phosphatidylserine externalization in hRBCs labelled with Annexin V FITC fluorescence (flow cytometry) (A–F) and the phagocytosis of these cells by mouse microglial cells (G–H).

<p>(A–F) PS externalization was (35.70%) most successful with incubation for 10 min with 20 µM NEM and then incubated with 20 µM PSox for 30 min. Although, 47.50% of hRBCs showed PS externalization after being incubated for 30 min with 30 µM NEM and then 30 min with 20 µM PSox for 30 min, cells were fragile and considered unsuitable for use. (G) Prelabled normal hRBCs were very rarely recognised by microglial cells, but (H) phagocytosis was common for prelabeled PSox-hRBS cells. (I) The hRBSCs phagocytosis rate was significantly higher for PSox-treated cells. Immunoflurescent staining indicated that the cultured cells expressed CD11b and MFG-E8 (J–L). (M) The majority of cultured microglial cells can incorporate microbeads (green) suggesting that they have the ability of non-specific phagocytosis. Scale bars: 50 µm for G–H, 100 µm for J–M. ** <i>P</i><0.01. Abbreviations: Con, control; PSox: mixture of oxidized and nonoxidized phosphatidylserine; hRBCs, human red blood cells.</p

Summary of the primers for qRT-PCR.

<p>Summary of the primers for qRT-PCR.</p

A murine glaucoma model induced by rapid <i>in vivo</i> photopolymerization of hyaluronic acid glycidyl methacrylate

<div><p>Glaucoma is an optic neuropathy commonly associated with elevated intraocular pressure (IOP) resulting in progressive loss of retinal ganglion cells (RGCs) and optic nerve degeneration, leading to blindness. New therapeutic approaches that better preserve the visual field by promoting survival and health of RGCs are highly needed since RGC death occurs despite good IOP control in glaucoma patients. We have developed a novel approach to reliably induce chronic IOP elevation in mouse using a photopolymerizable biomatrix, hyaluronic acid glycidyl methacrylate. This is achieved by rapid <i>in vivo</i> crosslinking of the biomatrix at the iridocorneal angle by a flash of ultraviolet A (UVA) light to impede the aqueous outflow pathway with a controllable manner. Sustained IOP elevation was induced after a single manipulation and was maintained at ~45% above baseline for >4 weeks. Significant thinning of the inner retina and ~35% reduction in RGCs and axons was noted within one month of IOP elevation. Optic nerve degeneration showed positive correlation with cumulative IOP elevation. Activation of astrocytes and microglia appeared to be an early event in response to IOP elevation preceding detectable RGC and axon loss. Attenuated glial reactivity was noted at later stage where significant RGC/axon loss had occurred suggesting astrocytes and microglia may play different roles over the course of glaucomatous degeneration. This novel murine glaucoma model is reproducible and displays cellular changes that recapitulate several pathophysiological features of glaucoma.</p></div

Ocular hypertension induced by HAMA xl led to significant loss of retinal ganglion cells (RGCs) one month post-operation.

<p>(A) Representative graphs of the Day 30 retinal flatmounts immunostained with BRN3A, a RGC nucleus marker. Loss of RGCs was observed from the hypertensive eyes treated with HAMA xl on Day 30. Note: the mouse monoclonal BRN3A antibody used in the present study cross-reacted with the blood vessels (red arrowheads) in the retina which became more visible in the degenerative retinas. Yellow dotted lines in b, c delineate the regions with more prominent RGC loss. Scale bar: 1 mm. (B) Representative micrographs of BRN3A immunolabeling of RGCs from normotensive (a. PBS+UVA light; b. HAMA monomer) and hypertensive (c. HAMA xl) retinas. Scale bar: 50 μm. (C) Quantification of RGC density based on BRN3A+ nuclei count from retinal flatmounts. Graph was shown as interleaved box & whiskers with 95% confidence interval. n = 11–12 eyes/group for naive, HAMA monomer (Day 30) and HAMA xl (Day 3) groups; n = 20 eyes/group for PBS+UVA light (Day 30) and HAMA xl (Day 30) groups, **** P<0.0001, ns: non-significant. Two-way ANOVA followed by Tukey’s multiple comparisons test. (D) Schematic indicating the sampling of eight 563μm x 422μm rectangle area in the retinal flatmount from four quadrants at two eccentricities (central vs periphery) from the optic nerve head (ONH) for RGC quantification (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196529#pone.0196529.s001" target="_blank">S1 Fig</a> for technical details).</p

Summary of existing surgically-induced rodent ocular hypertension models.

<p>Summary of existing surgically-induced rodent ocular hypertension models.</p

Astrocyte activation was observed in the hypertensive retinas with more prominent reactivity detected on Day 3 compared to Day 30.

<p>(A) Immunostaining of GFAP in retinal vertical sections. HAMA xl induced dramatic astrocyte activation with higher reactivity detected at early time point Day 3 (d) compared to Day 30 (e). In contrast, PBS+ UVA light (data not shown) or HAMA monomer (b,c) did not induce detectable astrocyte activation compared to naive controls. Images were collected from approximately 1.2–1.5mm from the optic nerve head in the retinal vertical sections. Scale bars: 50 μm. (B) Representative GFAP-immunoreactivity in retinal flatmounts. Top panel: GFAP immunostianing in retinal flatmounts from naive (f), hypertensive retina on day 3 (g) and day 30 (h); bottom panel: signals delineated by HALO software for corresponding images in f,g,h. No activation of astrocytes was noted in the retinal flatmounts from the PBS+UVA or HAMA monomer controls, data not shown. (C) Quantification of GFAP-immunoreactivity by HALO based on the total area covered by GFAP-immunofluorescence and the signal intensity. The intensity of GFAP-immunofluorescent signal was categorized as strong, moderate and weak by HALO. GFAP-immunoreactivity was significantly upregulated in the hypertensive retinas on Day 3 (P = 0.0027) and Day 30 (P = 0.0145) compared to naive controls. A significant attenuation of GFAP-immunoreactivity was also detected on Day 30 compared to Day 3 (P = 0.0036). Error bars: SEM. (D) GFAP mRNA was significantly upregulated in the hypertensive retinas on Day 3, which attenuated largely on Day 30. ** P = 0.008, multiple t-tests. Error bars: SEM.</p

In vivo photopolymerization of HAMA-μBeads induced sustained IOP elevation for over one month.

<p>(A) Schematic indicating the microinjection of HAMA-μBeads into the anterior chamber and photopolymerization of HAMA at the iridocorneal angle to impede the aqueous outflow. 1. An air bubble (1μl) was first injected into the central anterior chamber via an opening made at the paracentral cornea. 2. 2% HAMA-μBeads solution (2μl, indicated as blue here) was injected into the interface between the air bubble and the aqueous humor. The air bubble guided the distribution of HAMA-μBeads to the iridocorneal angle and prevented efflux of the solution upon removal of the micropipette. 3. Immediately post-injection, HAMA was photopolymerized by defined UVA light at 365nm wavelength for 10 seconds, the μBreads were immobilized within the solidified HAMA gel for long-term tracking of the morphological change of the gel inside the anterior chamber. (B) UVA lamp that was programmed to generate UVA light at 365nm for 10 seconds per action. (C) Representative anterior chamber images before and after injection. a. pre-injection, the blue dotted circle marks the limbal region where the iris joins the cornea and sclera; b. shows the HAMA-μBeads ring formed along the iridocorneal angle immediately after photopolymerization; c. shows the distribution pattern of the HAMA-μBeads inside the anterior chamber 30 days post-injection, the HAMA-μBeads gel remained in place at the angle after 1 month post-operation; d. a HAMA monomer injected eye 30 days post-injection. (D) Hematoxylin and eosin stain of ocular vertical sections at the iridocorneal angle from a naive eye (a) and a HAMA-μBeads injected eye (b), the blue matter between the iris and the cornea in (b) is polymerized HAMA. Red asterisks indicate the position of the schlemm’s canal. (E) IOP profiles from the control and HAMA xl groups. Injection of 1XPBS followed by the UVA exposure (the PBS+UVA light group), or injection of HAMA monomer without the presence of μBeads and UVA crosslink did not cause IOP elevation. In vivo photopolymerization of HAMA-μBeads induced significant and sustained IOP elevation. n = 10–12 eyes/group for naive and PBS+UVA light groups, n = 44 eyes/group for HAMA monomer and HAMA xl groups. Group comparison was performed by one-way ANOVA followed by Tukey’s multiple comparisons (F = 26.11, P<0.0001); IOP elevation in the HAMA xl group at each time point was compared with the PBS+UVA light group by Student’s T-test, * P<0.05, ** P<0.01, *** P<0.001. Error bars indicate 95% confidence interval.</p

List of primary antibodies used for this study.

<p>List of primary antibodies used for this study.</p