Inflammatory resolution and vascular barrier restoration after retinal ischemia reperfusion injury

Abstract

Background: Several retinal pathologies exhibit both inflammation and breakdown of the inner blood-retinal barrier (iBRB) resulting in vascular permeability, suggesting that treatments that trigger resolution of inflammation may also promote iBRB restoration.

Methods: Using the mouse retinal ischemia-reperfusion (IR) injury model, we followed the time course of neurodegeneration, inflammation, and iBRB disruption and repair to examine the relationship between resolution of inflammation and iBRB restoration and to determine if minocycline, a tetracycline derivative shown to reverse microglial activation, can hasten these processes.

Results: A 90-min ischemic insult followed by reperfusion in the retina induced cell apoptosis and inner retina thinning that progressed for approximately 2 weeks. IR increased vascular permeability within hours, which resolved between 3 and 4 weeks after injury. Increased vascular permeability coincided with alteration and loss of endothelial cell tight junction (TJ) protein content and disorganization of TJ protein complexes. Shunting of blood flow away from leaky vessels and dropout of leaky capillaries were eliminated as possible mechanisms for restoring the iBRB. Repletion of TJ protein contents occurred within 2 days after injury, long before restoration of the iBRB. In contrast, the eventual re-organization of TJ complexes at the cell border coincided with restoration of the barrier. A robust inflammatory response was evident a 1 day after IR and progressed to resolution over the 4-week time course. The inflammatory response included a rapid and transient infiltration of granulocytes and Ly6C⁺ classical inflammatory monocytes, a slow accumulation of Ly6C^neg monocyte/macrophages, and activation, proliferation, and mobilization of resident microglia. Extravasation of the majority of CD45⁺ leukocytes occurred from the superficial plexus. The presence of monocyte/macrophages and increased numbers of microglia were sustained until the iBRB was eventually restored. Intervention with minocycline to reverse microglial activation at 1 week after injury promoted early restoration of the iBRB coinciding with decreased expression of mRNAs for the microglial M1 markers TNF-α, IL-1β, and Ptgs2 (Cox-2) and increased expression of secreted serine protease inhibitor Serpina3n mRNA.

Conclusions: These results suggest that iBRB restoration occurs as TJ complexes are reorganized and that resolution of inflammation and restoration of the iBRB following retinal IR injury are functionally linked.

Keywords: Blood-retina barrier; Cox-2; Granulocytes; IL-1β; Ischemia-reperfusion injury; Leukocytes; Microglia; Minocycline; Monocytes; Myeloid-derived macrophages; Resolution of inflammation; Retinal vasculature; Serpina3n; TNF-α; Tight junctions.

Fig. 1

Retinal IR injury induced edema and neurodegeneration. A–D At the indicated times following IR injury, total retinal thickness and thickness of the inner and outer retinal layers were measured in situ using SD-OCT. Measurements were made at 4 compass points 350 μm from center of the optic nerve head and averaged. A Total retina spans from the inner limiting membrane (ILM) to the retinal pigment epithelium (RPE). B Inner retina spans from ILM to the OPL. C Outer retina spans from the OPL to the RPE. Note that the y-axis scales are equal in (B) and (C). A total of n = 30 eyes/group were initiated at time zero but, due to cataract formation in some injured eyes, IR group size declined to 15 eyes/group at day 28. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 for IR versus sham by t test. D At the indicated times following IR injury, ongoing cell death was evaluated in Sham and IR-injured retinas using a DNA fragmentation assay with n = 6–8/group. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 for IR versus Sham by t test. E TUNEL staining and IHC of the RGC marker RBPMS was performed on flat-mounted Sham and IR retinas harvested at 1 day following injury

Fig. 2

Retinal IR injury caused breakdown of the iBRB that resolved after 2 weeks. A At the indicated times following IR injury, vascular permeability in Sham and IR retinas was evaluating by measuring the FITC-BSA accumulation into retinal tissue with n = 8/group: **p ≤ 0.01. B Representative images of permeability to sulfo-NHS-biotin in the superficial retinal vasculature (top panels) and the deep retinal vasculature (lower panels) obtained by confocal microscopy of Sham and IR retinas at 24 h after injury. Images show IF detection of the endothelial marker CD31 (PECAM-1, green) and the TJ protein ZO-1 (magenta), together with intravenously injected sulfo-NHS-Biotin (red) as an indicator of leakage from the vasculature. Note areas of sulfo-NHS-biotin leak corresponding to disruption of ZO-1 localization at endothelial cell borders

Fig. 3

Restoration of the vascular barrier following IR injury did not coincide with diminished perfused capillary volume or loss of vessel endothelialization. A Mice were perfused with FITC-BSA and immuno-probed for CD31 (PECAM-1) and ColIV followed by flat mounting and confocal microscopy. Arrows indicate empty sleeves that are positive for ColIV but lack FITC-BSA perfusion and CD31. B–D Percent of FITC-BSA and CD31 co-localized with ColIV was determined at 2 day (B), 2 weeks (C), and 4 weeks (D) after IR injury. (b–d) Density of ColIV-positive, FITC-negative, and CD31-negative empty sleeves were determined at 2 days (b), 2 weeks (c), and 4 weeks (d) after IR injury. No significant differences were observed between Sham and IR groups using both parametric (t test) and non-parametric (u test) statistics

Fig. 4

Restoration of the vascular barrier coincided with reorganization of TJ complexes. A At the indicated times following IR injury, retinas were removed and proteins in whole retinal lysates examined by Western blotting with antibodies to occludin phosphorylated on serine 490 (pS490), total occludin protein, ZO-1, ZO-2, and claudin-5. A representative blot is shown. B Quantification of 4 replicate Western blot experiments. Occludin pS490 immunoreactivity was normalized to that of total occludin. ZO-1, ZO-2, and claudin-5 immunoreactivities were normalized to that of β-actin. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by t test. C At various times following IR injury (2-day and 4-week samples shown), retinas were removed and immuno-probed with antibodies to occludin and ZO-1, followed by flat mounting and confocal microscopy. Arrows indicate discontinuities in TJ proteins at endothelial cell borders. D Histograms represent the evaluation of TJ continuity at endothelial cell borders using a blinded rank (1–5) scoring system. In the graph, green indicates completely continuous, yellow indicates 75% to 100% continuous, pink indicates 50% to 75% continuous, orange indicates 25% to 50% continuous, and red indicates 0% to 25% continuous border staining. For each retina, four images equidistant from the optic disc were averaged with n = 4 retinas for each condition. *p ≤ 0.05, ****p ≤ 0.0001 by t test

Fig. 5

IR injury induced progressive changes in innate immune cell populations within the retina. A Representative scatter-graphs showing the flow-cytometric analysis used to quantify immune cell populations in the retina. After gating for single cells, events were gated into CD11b⁺/CD45^low (principally microglia), CD11b⁺/CD45^hi myeloid leukocytes, and CD11b^neg/CD45^hi lymphocytes. Myeloid leukocytes were further gated into CD11b⁺/CD45^hi/Ly6C^hi/Ly6G^neg classical monocytes, CD11b⁺/CD45^hi/Ly6C^neg/Ly6G^neg non-classical monocytes or MDM, and CD11b⁺/CD45^hi/Ly6C⁺/Ly6G⁺ granulocytes. Microglia-like cells were further gated to quantify CD11b⁺/CD45^low/Ly6C^neg/Ly6G^neg microglia. B–E At the indicated times following IR injury, flow-cytometric analysis was used to quantify microglia and leukocyte populations in Sham and IR-injured retinas, including: granulocytes (B), classical monocytes (C), non-classical monocytes/MDM (D), and microglia (E). For each analysis, 4 retinas were pooled and analyzed with n = 4 pools of retinas for each group at 1 day, 4 days, 1 week, and 4 weeks following IR injury. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 by one-way ANOVA with Bonferroni and Sidak multiple comparison test. F Representative images obtained by confocal microscopy of the superficial retinal vasculature of at the optic nerve head and periphery of Sham and IR retinas at 24 h after injury. Images show IB4 (green, note: binds to endothelium, with arterial > venus, and to some leukocytes), ColIV (red), and CD45 (magenta). Note that CD45⁺ leukocytes appear both within vessel lumen and within the retina. G Confocal microscopy images showing IB4 (green), ZO-1 (red), and CD45 (magenta) of a superficial vascular region of an IR-injured retina at 24 h after injury (Sham not shown). Arrows indicate regions where the vessel is exhibiting disorganization of endothelial TJ complexes coinciding with apparent extravasation of CD45⁺ leukocytes

Fig. 6

Retinal tissue invasion of CD45⁺ leukocytes following ischemia-reperfusion (IR). Mice eyes were subjected to IR for 90 min or needle puncture only (Sham), followed by natural reperfusion. At 1 day (1d) or 2 days (2d) after IR, mouse retinal whole mounts were analyzed for A IF for CD45 (green) and IB4 (magenta) at both the superficial vascular plexus (left panel) and deep vascular plexus (right panel) of retinal whole mounts from IR retinal at 1 d after reperfusion. Scale bars: 40 μm. B Imaris software processed images showing 3D surface re-construction for identifying the vessels stained with IB4 (magenta) and the number of CD45⁺ cells (green with grey dots). Scale bars: 40 μm. C Representative Imaris software 3D re-constructed image (left panel) showing the location of luminal (yellow) and extravascular (green) CD45⁺ cells in relation to the IB4-positive vessels (magenta). The magnified portion of the boxed region is shown on the right panel. Scale bars: 10 μm (left panel) and 2 μm (right panel). D Results of quantification of CD45⁺ cell localization in relation to IB4⁺ vessels in the superficial vascular plexus. E Quantification of CD45⁺ cell localization in relation to IB4 stained vessels in the deep vascular plexus. Cell numbers were normalized to the calculated volume (mm³) of IB4⁺ vascular plexus in each image to obtain relative cell densities. For each condition, two representative image fields from each retina of Sham (n = 1) and IR (n = 3) were quantified

Fig. 7

Retinal IR injury caused microglia proliferation and mobilization toward the GCL. A Representative images of IF of Iba-1 (microglia/phagocyte marker) and Ki-67 (proliferative cell marker) in retinal sections from Sham and IR retinas at 2 days following IR injury. B Representative confocal microscopic images of IB4, Iba-1, and Ki-67 IF in flat-mounted retinas from naive (no treatment), Sham-treated, and IR-injured retinas. Z-stacks of images from retinal layers containing the superficial vascular plexus (GCL and nerve fiber layer) and the deep vascular plexus (IPL, INL, and OPL). C Quantification of microglia and proliferative cell densities in retinal layers. Cell counts from confocal microscopic images (as shown in A, 4 images/retina/depth) were used to quantify Iba-1⁺ and Ki-67⁺ cell densities (cells per retinal area) and the extent of co-localization of these markers with n = 2 retinas/group for naive and n = 4 retinas/group for Sham and IR and *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 for IR versus Sham by one-way ANOVA. D Effect of IR injury on microglia/phagocyte activation evaluated by increase in soma size, an indication of loss of process ramification, and/or an amoeboid morphology. Confocal microscopic images of Iba-1 IF in (B) were used to evaluate soma sizes (areas) of Iba-1+ microglia/phagocytes. Note that Iba-1⁺ cells in IR-injured retinas had significantly larger mean soma size than both naive and Sham retinas. *p < 0.05, ***p < 0.001 by one-way ANOVA

Fig. 8

Lineage tracing of microglia show their increase in the inner retina and appearance of CD11b⁺/Iba-1⁺ myeloid leukocytes after IR injury. CX3CR1-CreERT2 knockin driver mice crossed with mT/mG reporter mice were used to lineage trace microglia as GFP⁺. A 4-week washout period after TAM treatment was used to clear GFP⁺ Cre-recombined monocytes from circulation prior to IR injury. A Representative images of GFP, CD11b, and Iba-1 expressing cells in inner (GCL + IPL) and deep (INL + OPL) layers of Sham and IR-injured retinas at 4 days after injury. Note that essentially all CD11b⁺ cells in the Sham retina are GFP⁺ microglia expressing Iba-1. Quantification and categorization of CD11b⁺ cell densities (cells per retinal area) in the inner layers (GCL + IPL) (B), deep layers (INL + OPL) (C), and combined layers (GCL + IPL + INL + OPL) (D). Note that the density of CD11b⁺/Iba-1⁺/GFP⁺ microglia nearly doubles in the inner layers, while not changing in the deep layers. An appreciable population of CD11b⁺/Iba-1⁺/GFP^neg myeloid leukocytes appears in both layers after IR injury, while there are relatively few myeloid leukocytes that are negative for Iba-1. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 for IR versus Sham by u test

Fig. 9

Iba-1⁺ phagocytes express CD68 and engulf cells in the GCL after IR injury. A Representative confocal microscope images of retinal sections from a Sham retina at 4 days after IR injury showing nuclei (Hoechst staining), microglia/phagocytes (Iba-1), Brn3a (a nuclear protein expressed in a major subset of RGC), and CD68 (a marker of phagocytes). Note that in the Sham retina there are few microglia/phagocytes in the GCL and essentially no detectable CD68. B Representative image of IR-injured retina section at 4 days suggesting mobilization of Iba-1⁺ microglia and close association of Iba-1⁺/CD68⁺ phagocytes with nuclei in the GCL. C Three-dimensional reconstructions using Imaris software showing a Iba-1⁺ microglia/phagocyte engulfing a Brn3a⁺ RGC in the GCL of an IR retina. The left image shows nuclei (blue), Iba-1 (green), and CD68 (magenta). The right image shows Brn3a (red), Iba-1 (green), and CD68 (magenta)

Fig. 10

Intervention treatment with minocycline promotes restoration of the iBRB. Mice were treated by daily injections of minocycline (50 μg/g-BW i.p.) or saline vehicle beginning 7 days after retinal IR injury. At 14 days following IR injury, vascular permeability in Sham and IR retinas was evaluating by measuring the accumulation of intravenously injected FITC-BSA in retinal tissues. n = 13–16/group. ***p ≤ 0.001 by two-way ANOVA with Tukey post-hoc test

Fig. 11

Effect of intervention treatment with minocycline on retinal inflammatory gene expression. Mice were treated by daily injections of minocycline (50 μg/g-BW i.p.) or saline vehicle beginning 7 days after retinal IR injury. At 10 days following IR injury, retinas were harvested and total RNA isolated. Duplex qRT-PCR, with Actb mRNA as reference, was used to determine levels of Tnfa (A), Il1b (B), Ptgs2 (C), Cd68 (D), Cyba (E), Cybb (F), Lcn2 (G), Serpina3n (H), Arg1 (I), Cd200r1 (J), Tgm2 (K), and Mrc1 (L) mRNAs in whole retinas. n = 8/group. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by two-way ANOVA with Tukey post-hoc test