Journal of Neuroinflammation

Zhou et al. Journal of Neuroinflammation(2024) 21:210
https://doi.org/10.1186/s12974-024-03190-9Open Access

RESEARCH

Suppression of inner blood-retinal barrier
breakdown and pathogenic Müller glia
activation in ischemia retinopathy by myeloid
cell depletion
Lingli Zhou1,2, Zhenhua Xu2, Haining Lu2, Hongkwan Cho2, Yangyiran Xie2, Grace Lee2, Kaoru Ri2 and Elia J. Duh2*
Abstract
Ischemic retinopathies including diabetic retinopathy are major causes of vision loss. Inner blood-retinal barrier(BRB) breakdown with retinal vascular hyperpermeability results in macular edema. Although dysfunction of theneurovascular unit including neurons, glia, and vascular cells is now understood to underlie this process, there is aneed for fuller elucidation of the underlying events in BRB dysfunction in ischemic disease, including a systematic
analysis of myeloid cells and exploration of cellular cross-talk. We used an approach for microglia depletion with
the CSF-1R inhibitor PLX5622 (PLX) in the retinal ischemia-reperfusion (IR) model. Under non-IR conditions, PLX
treatment successfully depleted microglia in the retina. PLX suppressed the microglial activation response following
IR as well as infiltration of monocyte-derived macrophages. This occurred in association with reduction of retinal
expression of chemokines including CCL2 and the inflammatory adhesion molecule ICAM-1. In addition, there wasa marked suppression of retinal neuroinflammation with reduction in expression of IL-1b, IL-6, Ptgs2, TNF-a, andAngpt2, a protein that regulates BRB permeability. PLX treatment significantly suppressed inner BRB breakdown
following IR, without an appreciable effect on neuronal dysfunction. A translatomic analysis of Müller glial-specificgene expression in vivo using the Ribotag approach demonstrated a strong suppression of Müller cell expressionof multiple pro-inflammatory genes following PLX treatment. Co-culture studies of Müller cells and microglia
demonstrated that activated microglia directly upregulates Müller cell-expression of these inflammatory genes,
indicating Müller cells as a downstream effector of myeloid cells in retinal IR. Co-culture studies of these two cell
types with endothelial cells demonstrated the ability of both activated microglia and Müller cells to compromise
EC barrier function. Interestingly, quiescent Müller cells enhanced EC barrier function in this co-culture system.
Together this demonstrates a pivotal role for myeloid cells in inner BRB breakdown in the setting of ischemiaassociated disease and indicates that myeloid cells play a major role in iBRB dysregulation, through direct andindirect effects, while Müller glia participate in amplifying the neuroinflammatory effect of myeloid cells.
Keywords Microglia, Macrophages, Csf1r, Müller glia, Blood-retinal barrier, Neuroinflammation*Correspondence:
Elia J. Duh
eduh@jhmi.edu

1State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center,
Guangdong Provincial Key Laboratory of Ophthalmology and Visual
Science, Sun Yat-Sen University, Guangzhou, China
2
Wilmer Eye Institute, Johns Hopkins University School of Medicine,
Baltimore, MD, USA© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Zhou et al. Journal of Neuroinflammation(2024) 21:210

Background
Ischemic retinopathies including diabetic retinopathy (DR) and retinal vein occlusions (RVO) are major
causes of vision loss globally [1, 2]. The most common
cause of visual impairment in these conditions is macular edema, a result of increased retinal vascular permeability that leads to intraretinal and subretinal fluid [1–3].
Although anti-VEGF agents have led to improvements invisual outcomes, many patients do not fully respond totreatment. In addition, many patients require continued
treatment over many years for both conditions, resulting
in a large treatment burden [1, 2]. There is a great needfor improved understanding of the causes and mechanisms regulating retinal vascular permeability, in order toenable development of additional treatments.
The blood-retinal barrier (BRB) plays an integral rolein homeostatic maintenance of the retinal microenvironment through its selective regulation of flux of moleculesfrom the systemic circulation [4, 5]. The BRB bears great
similarity to the blood-brain barrier in its characteristics
and critical function in regulating entry of blood-borne
substances, including potentially harmful molecules, into
the neural parenchyma [4]. The blood-retinal barrier has
two distinct barrier systems, the inner and outer bloodretinal barriers [6, 7]. The inner blood-retinal barrier is
primarily maintained by tight junctions of endothelial
cells lining the retinal blood vessels, analogous to cerebral ECs of the blood-brain barrier [4, 5] and are criticalto the health and integrity of the inner retina. The outerblood-retinal barrier (oBRB) is maintained by the retinal
pigment epithelium (RPE), which regulates the transportof molecules between the choriocapillaris and the deep
(outer) retina [6]. Integrity of the outer BRB is known
to be critical, especially for diseases of the outer retina
including age-related macular degeneration.
Breakdown of the inner BRB is a pivotal event in macular edema, the most common cause of visual impairment
in ischemic retinopathies including DR and RVO [1, 3,
5]. Although compromise of retinal vascular endothelial
cells, including its tight junctions, is recognized to be a
critical proximal cause of vascular hyperpermeability, it
is increasingly recognized that the pathophysiology of
BRB dysfunction involves a broader breakdown of theneurovascular unit (NVU) [4, 8]. The cellular constituents of the retinal NVU include neurons and glial elements (Müller glia and astrocytes) in addition to vascular
cells. Consequently, extensive research efforts are being
dedicated to understanding the interactions between thecellular constituents of the NVU, in order to gain insights
into mechanisms governing BRB health and disease.
Mononuclear phagocytes (MPs), or myeloid cells, havedrawn increased attention as regulators of ischemic retinopathies and the retinal neurovascular unit [7, 9, 10]. In
models of ischemia-induced retinal angiogenesis, MPs

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play a role in promoting pathologic neovessel formation [7, 11, 12]. With respect to the retina, MPs primarily
comprise resident microglia and monocyte-derived macrophages from the systemic circulation [13]. Microglia
are the principal resident immune cells of the retina andbrain [14]. Although these cells play an important role indevelopment and homeostatic maintenance, their dysregulation in disease contexts can result in retinal tissue
damage and pathology, in part by recruiting monocytederived macrophages from the systemic circulation [9,13, 14].
In order to gain insights into their potential pathogenicrole in both systemic and ocular diseases, microglial
depletion strategies have been of great interest, to gain
insights into their role. A particularly useful approachfor depletion has been pharmacologic inhibition of CSF1R, an important survival factor for microglia. The agent
PLX5622 (PLX) has been especially valuable, given its
high selectivity for CSF-1R, excellent penetration into the
CNS, and high efficiency of microglia depletion [15]. In
part due to its ability to penetrate the blood-brain barrier,
PLX has been used to gain insights into the role of bothneurodegenerative and neuroinflammatory CNS diseases
including Alzheimer [16] and experimental autoimmune
encephalitis, a mouse model of multiple sclerosis [17,18]. Similarly, this agent has been immensely useful forstudying microglia in diverse retinal conditions including
retinal degenerations and uveitis [19, 20]. More recent
studies indicate that PLX also impacts function of systemic macrophages, especially with longer term treatment, so it is conceivable that the phenotypic effects of
PLX treatment may be due at least in part on macrophages as well as microglia [21].
CSF-1R inhibition with PLX has been used to implicate
the role of microglia/macrophages in the outer bloodretinal barrier, in a mouse model of endotoxin-induced
uveitis (EIU) [22]. In this model, in which mice were
subjected to repeated, systemic challenge with lipopolysaccharide (LPS), the authors demonstrated prominent
subretinal fluid indicative of breakdown of the outerblood-retinal barrier. This was accompanied by prominent localization of Iba1 + cells in the subretinal space (inproximity to the subretinal fluid) and in the distal outer
nuclear layer. CSF-1R inhibition suppressed the oBRB
breakdown and subretinal fluid compared to vehicle.
Notably, CSF-1R inhibition has not yet been reported
in regard to inner BRB breakdown and ischemic retinal
diseases.
Oxidative stress and inflammation are known to be
critical drivers of disease progression of ischemic retinopathies [23]. Neuroinflammation is an importantelement of ischemic retinopathies. The mouse modelof retinal ischemia-reperfusion is a model of ischemic injury involving the inner retina [24]. It involves

Zhou et al. Journal of Neuroinflammation(2024) 21:210

pathophysiologic processes including oxidative stress andneuroinflammation in common with ischemic retinopathies including diabetic retinopathy. This model has been
useful to study mechanisms of neurodegeneration [24]and inner blood-retinal barrier breakdown [25, 26]. In
this study, we further characterized immune cell activation of mononuclear phagocytes in retinal IR and studied
the impact of PLX treatment. Our depletion studies support the pivotal role of microglia/macrophages in inner
blood-retinal barrier breakdown associated with retinal
ischemia, in part by reprogramming of the Müller glia
population.

Materials and methods
Animals

Animal studies were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins
University School of Medicine. All procedures involving animals were conducted under the Association for
Research in Vision and Ophthalmology Statement for
the Use of Animals in Ophthalmic and Vision Research.
C57BL/6J mice, Glast-Cre-ERT(#012586) and RiboTag(B6J.129(Cg)-Rpl22tm1.1Psam/SjJ; catalog 029977) mice
were purchased from the Jackson Laboratory. The animals were housed at 20–23 °C under 12:12 h light/darkcycles.
Myeloid cell depletion with PLX5622

The CSF1R inhibitor PLX5622, which was provided
by Plexxikon Inc. (USA), was formulated in AIN-75 Astandard chow by Research Diets (USA). AIN-74 A
standard chow served as a vehicle. For all retinal ischemia-reperfusion experiments, 8 week-old mice were fed
with PLX5622 or vehicle diet starting at 7 days before
retinal ischemia-reperfusion injury until each respective
endpoint.
Tamoxifen treatment

Tamoxifen was administrated as previously described[27]. In brief, tamoxifen (sigma T5648) was dissolvedin corn oil (Sigma, USA, C8267) at a concentration of
7 mg/mL. Mice at P21 received a 3-day injection of 1 mg
of tamoxifen and another 2-day injection of 2 mg oftamoxifen.
Mouse model of ischemia-reperfusion (IR)

Mouse IR was performed as previously described [28].
In brief, mice were anesthetized with a cocktail of ketamine, xylazine, and acepromazine in PBS for 5–10 min
to achieve deep anesthesia. A 30-gauge cannula needle
attached to a line infusing sterile saline was inserted
into the anterior chamber of the mice. Retinal ischemia
was induced by increasing the intraocular pressure
to 110 mmHg by raising the height of the sterile saline

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bottle. Ischemia was confirmed when the retina turned
pale. After 90 min, the needle was removed. Mice wereplaced on a heating pad until they recovered from theanesthesia.
Flow cytometry analysis

Flow cytometry was done as previously described [29].
Single-cell suspensions from the retina was obtainedwith the Neural Tissue Dissociation Kit (Miltenyi Biotec, US;130-093-231) and stained with APC anti-CD11b(eBioscience), FITC anti-CD45 (eBioscience), PE-Cy7
anti-CD86 (BD Biosciences), BV421 anti-CD206 (BioLegend), BUV 496 anti-CD192 (BD Biosciences), PE antiLy6G (BD Biosciences) and BUV 395 anti-CD11c (BD
Biosciences) for 30 min. A 4-laser BD LSR II (BD Biosciences) was used to collect the data, and FlowJo software(BD Biosciences) was used for analysis. The gating strategy is presented in Fig. S1.
Ribosome immunoprecipitation

Isolation of polysome-bound mRNA using RiboTag fromtissues was performed as previously described ( [29]).
After homogenization in 400 µL of polysome buffer(50 mM Tris, pH 7.5, 100 mM KCl, 12 mM MgCl2, 1%
NP-40, 1 mM DTT, 200 U/mL RNasin, 1 mg/mL heparin, 100 µg/mL cycloheximide, and 1× protease inhibitor)
using pellet pestles (Kimble Chase), retinal homogenates
were centrifuged at 15,300 g for 10 min at 4 °C. 10 µl of
the supernatant was collected for input. The rest of thesupernatant was incubated with anti-HA antibodyconjugated magnetic beads (M180-11; MBL International) at 4 °C overnight on an orbital shaker. The nextday, the beads were washed 4 times with high-salt buffer (50 mM Tris, pH7.5, 300 mM KCl, 12 mM MgCl2,
1% NP-40, 1 mM DTT, and 100 µg/mL cycloheximide).
Beads were then resuspended in 350 µL RLT buffer plus
β-mercaptoethanol, thoroughly vortexed, and pelletedusing a magnetic separator. The supernatant was transferred into a new tube for RNA extraction with RNeasy
Micro kit (QIAGEN). cDNA was synthesized withLunaScript® RT SuperMix Kit (NEB #M3010).
Real-time PCR analysis for retina tissue

RNeasy mini kit (QIAGEN) was used for RNA isolationfrom total retinas and cultured cells. Single-stranded
cDNA was synthesized using M-MLV Reverse Transcriptase (Invitrogen) as previously described (59). Quantitative PCR was performed with SYBR Green PCR MasterMix (Invitrogen) with the StepOnePlus real-time PCRsystem (Applied Biosystems). Gapdh was used fornormalization.

Zhou et al. Journal of Neuroinflammation(2024) 21:210

Immunofluorescent staining

After fixation with 4% PFA and dehydration with 30%
sucrose, eyes were embedded in an OCT compound
solution. The cryo-blocks were sectioned using a cryomicrotome (10 μm sections) along the optic nerve. The
sections were blocked with donkey serum and thenstained with mouse anti-rabbit Iba1 (Wako), goat antimouse albumin (Novus), and rat anti-mouse CD31 (BD
Biosciences) at 4 °C overnight. After washing 3 times, thesections were then incubated with secondary antibodies
and DAPI. Photographs were taken using a Zeiss LSM
710.
Leukostasis assay

Mice were deeply anesthetized and perfused with 37 °C
of PBS for 3 min for removal of all nonadherent cells.
Mice were then perfused over a span of 2–3 min with
5 ml rhodamine-labeled Concanavalin A (ConA; 40 µg/
ml in PBS; Vector Labs) followed by 10 mL of PBS for
4 min. Eyeballs were then fixed in 4% PFA. The retinas
were flat-mounted and visualized under confocal Zeiss
LSM 710.
Blood-retinal barrier (BRB) permeability assay

The blood-retinal barrier (BRB) assay was performed as
described [30]. Mice were given an intraperitoneal injection of 1 µCi/1 g of [3H]mannitol (PerkinElmer, US).
Retinas and part of the lung were collected and weighed
1 h after the injection. 1 mL NCSII solubilizing solution
was added to each retina and lung tissue and incubated
at 50 °C overnight. The next day, solubilized tissue wasbrought to room temperature and decolorized with 20%
benzoyl peroxide in toluene at 50 °C. After returning toroom temperature, 5 mL of scintillation fluid and 30 µL
Glacial Acetic Acid were added to each vial. The samples
were then stored at 4 °C overnight in the dark to eliminate chemoluminescence. Radioactivity was countedwith a scintillation counter (LS 6500 Liquid Scintillation
Counter; Beckman-Coulter, Indianapolis, IN, USA). The
cpm of retina tissue and lung tissue were measured andnormalized to the dry weight of each sample.
Flash scotopic electroretinography (ERG)

ERG was performed as previously reported [31]. Afterdark-adaptation overnight, mice were anesthetized withketamine (100 mg/kg) and xylazine (10 mg/kg), andpupils were dilated with topical tropicamide (1%). Scotopic ERG responses were measured using the Celeris ERG
stimulator (Diagnosys, Lowell, MA) at flash intensities
of 0.025, 0.250, 2.500, 7.900, and 79.050 cd*s/m2. ERGs
were measured simultaneously from both eyes with aground electrode placed into the forehead between theeyes and a reference electrode into the hip. 10 traces
were averaged for the intensity of 0.025,0.250, 2.500, andPage 4 of 16

7.900 cd*s/m2, and 6 traces were averaged for the intensity of 79.050 cd*s/m2.
Cell culture

Primary mouse Müller cells were isolated and cultured
as previously described [32]. Eyeballs from C57BL/6J
mice at P5 to P7 were removed and placed in serumfree DMEM (Thermo Fisher Scientific) plus 1× GlutaMAX supplement (Thermo Fisher Scientific) overnightat room temperature in the dark. The eyeballs were thenincubated in a buffer containing trypsin, EDTA, and collagenase I at 37 °C. Retinas were dissected and placed in
DMEM (high glucose) supplemented with 10% FBS, and
1× GlutaMAX. Retinas were minced into small pieces
using forceps and incubated in the cell culture incubator for 3–4 days. Cultures were washed vigorously withthe medium until only flat and firm adherent cell populations remained. Primary Müller cells were characterized
by immunostaining with the Müller cell-specific markers glutamine synthetase (GS) and vimentin. Müller cells
were used from passages 3 to 5.
BV2 cells were maintained in DMEM/F12 culture
medium with 10% FBS. For the BV2 and Müller cell coculture experiment, BV2 cells were seeded at a densityof 0.5 × 10^5 on the Transwell inserts with a pore size of
0.4 μm (Corning #3470). After starvation with DMEM/
F12 without FBS, the BV2 cells were treated with or without LPS (1 µg/ml) for 6 h. After rinsing with DMEM/
F12 for 3 times, the inserts were then placed on the wells
seeded with Müller cells and cultured for 48 h. After 48 h,
the inserts were removed. The Müller cells were collected
for RNA extraction.
Endothelial cell solute flux assay

Endothelial cell solute flux assay was performed as previously described [33]. Human umbilical vein endothelial
cells (HUVECs) were maintained in EGM2-MV culture
medium (Lonza). For the solute flux assay, HUVECs wereseeded at a density of 3 × 104 on Transwell inserts (poresize 0.4 μm Corning #3470) which were pre-coated withfibronectin (Sigma). Once they were confluent, HUVECs
were treated with EBM2 with 0.5% FBS overnight for
starvation.
For the BV2 and Müller cell co-culture experiment,
BV2 cells were seeded at a density of 0.5 × 105 on Transwell inserts with a pore size of 0.4 μm (Corning #3470).
BV2 cells were treated with or without LPS (1 µg/ml)
for 24 h. After rinsing three times with DMEM/F12, theinserts were then placed over wells seeded with Müller
cells in DMEM with 5% FBS and cultured for 72 h. After
72 h, inserts with BV2 were replaced with inserts with
HUVECs and co-cultured for another 24 h. One insert
without HUVECs served as blank control. 50 µl of 100µM RITC-Dextran (Mol wt ~ 70,000; #9379, Sigma) wereZhou et al. Journal of Neuroinflammation(2024) 21:210

applied to the top inserts. 50 µl samples from the bottom chamber were collected at 30, 60, 90, 120,150, and
180 min. Additional 50 µl samples were collected fromthe inserts at 180 min. The fluorescein content of eachsample was measured with FLUOstar OPTIMA (BMG
Labtech GmbH). The diffusive permeability was calculated as previously reported [33].
Statistical analysis

All bar graphs represent mean ± standard error (SEM).
Unpaired, 2-tailed Student’s t-test was used for statisticalanalysis between 2 groups. One-way ANOVA was used
to perform statistical analysis between 3 groups. Twoway ANOVA was used to perform statistical analysis
between multiple groups of different treatments. Analysis was performed using GraphPad Prism, version 10.
p < 0.05 was considered statistically significant.

Results
Myeloid cell activation and infiltration in the retinal
ischemia-reperfusion (IR) model

The rodent model of retinal ischemia-reperfusion has
been a valuable model for gaining insights into ischemic
retinopathies including diabetic retinopathy and vein
occlusions [34, 35]. The pathophysiology of this model
involves both oxidative stress and inflammation, exhibiting highly relevant pathology including blood-retinal
barrier breakdown [25, 36] and neurodegeneration[37]. Previous studies have demonstrated the activation of immune cells in the IR model [38]. In this study,
we further investigated immune cell activation in retinal IR. Using flow cytometric analysis, we observed anincreased number of Ly6G-CD45+CD11b+ myeloid cells
in the retina 3 days after IR injury, which constitutes both
microglia and macrophages (Fig. 1A). Although CD45
expression can differentiate these two cell populations in
non-disease settings, a recent study found that expression of CD45 in resident microglia cells was upregulated
upon activation [39], limiting its use in differentiation of
MPs in disease.
We further examined the expression of markers of
myeloid cell activation and peripheral immune cell
infiltration (Fig. 1B-E). We found an increased expression of CD206 and CD86 on the Ly6G-CD11b+ CD45+
cells. CD206 and CD86 were previously used as markers to indicate microglia/macrophage activation andpolarization. The increased expression of CD206 andCD86 may indicate an increased activity of endocytosis/phagocytosis of the myeloid cells [40, 41]. We also
observed increased expression of CD192, also known as
CCR2, on the myeloid cells. CCR2 is expressed predominantly in the monocytes [42]. This finding suggests thatthe increased number of CD11b+ and CD45+ cells weobserved includes infiltrating cells of monocytic origin.

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It should be noted that CCR2+ monocytes downregulate CCR2 expression after they migrate to the retina anddifferentiate into macrophages, making it difficult todistinguish the relative contributions of microglia andmonocyte-derived macrophages to the mononuclear
phagocyte population. Finally, we observed increased
expression of CD11c. A previous study found that a subset of microglia responsible for phagocytic and proinflammatory responses expressed CD11c [43]. Overall,these findings suggest a significant activation of resident
microglia as well as infiltration of peripheral myeloid cells
into the retina after IR injury.
CSR-1R inhibitor PLX5622 treatment reduced myeloid cellnumber and chemokine expression

In order to investigate the role of myeloid cells, we useda microglia depletion strategy with PLX5622 (PLX), a
Colony-Stimulating Factor 1 Receptor (CSF1-R) inhibitor. As shown in Fig. 2A, mice were administered a dietwith PLX or control vehicle (Vehicle) beginning 7 days
before IR. After 3 days of IR, retinas were collected andanalyzed with flow cytometry. The contralateral eyes withno IR injury served as NIR control. We first confirmed
the efficiency of myeloid cell depletion with PLX in the
NIR control eyes. As shown in Fig. 2B, there was a significant reduction of the Ly6G-CD11b+ CD45+ cells in
retinas from PLX-treated mice. We then investigated thechange in myeloid cell number in the IR eyes. As shownin Fig. 2B, we found that there was a significant reductionof the Ly6G-CD11b+CD45+ myeloid cells in the PLXtreated IR group compared to the CN-treated IR retinas.
In addition, there was no difference in the myeloid cellnumber between the PLX-NIR and the PLX-IR groups.
These findings suggest that inhibition with CSF-1R inhibitor PLX significantly depleted the myeloid cells, including both resident microglia and peripheral myeloid cells,in the retina after IR. In addition to the primary depletionof microglia, this effect of PLX treatment could be due
either to a reduction in number of monocytes available toinfiltrate into the retina or to the lack of microglia-mediated recruitment of systemic monocytes. Of note, we did
not observe a change in monocyte number in spleenscollected from the PLX-treated mice (Fig. S5), suggestingthat the latter explanation may be more important cause.
Since there was a reduction of peripheral myeloid cell
infiltration with the PLX treatment, we next looked into
the expression change of chemokines and adhesion molecules in retinas with PLX treatment after IR. As expected,
I/R induced a significant increase in the expression ofchemokines including Ccl2, Cxcl2, and Cxcl10 and theadhesion molecule Icam1 after 16 h of IR. PLX treatment significantly reduced the expression of these genes
in the retina after IR (Fig. 2C). This finding suggests thatthe depletion of myeloid cells led to a reduction of both

Zhou et al. Journal of Neuroinflammation(2024) 21:210

Page 6 of 16

Fig. 1 Myeloid cell infiltration and activation in the retinal ischemia-reperfusion model. A Flow cytometric analysis demonstrated increased Ly6G− CD45+
CD11b+ myeloid cell numbers in the retina after 3 days of IR. B Increased expression of CD206, CD86, CD192, and CD11c on the Ly6G− CD45+ CD11b+
myeloid cells at 3 days after IR. Red curve: IR; blue curve: NIR; grey curve: unstained blank control. Data are shown as mean ± SEM, n = 6–9 mice per group,
where each dot represents an individual eye. *p < 0.05 using Student’s t test

chemokine and adhesion molecule expression in the retina, thereby ameliorating inflammatory cell infiltration
into the retina.
We next performed a leukocyte adhesion assay at
3 days after IR. As shown in Fig. 2D, IR resulted in a
dramatic increase in leukocyte adhesion, which wasobserved in blood vessels from both the central andperipheral retina. PLX treatment significantly suppressed
IR-induced leukostasis.

Myeloid cell depletion ameliorated retinal
neuroinflammation induced by IR

We studied the effect of myeloid cell depletion with PLX
on retinal gene expression in IR. We first investigated theexpression of inflammatory cytokines in the total retina
after IR. At 16 h after IR, there was a significant increasein the expression of multiple inflammatory genes, including Il1b, Il6, Ptgs2, Sphk1, and Tnf. PLX-treated retinas exhibited significant reduction in the expression ofIl1b, Il6, Ptgs2, Sphk1, and Mmp9 compared to the CNtreated retinas. Interestingly, we only observed a trend of
reduction of Tnf in the PLX-treated retinas after IR. This

Zhou et al. Journal of Neuroinflammation(2024) 21:210

Page 7 of 16

Fig. 2 Depletion of myeloid cells in IR ameliorates inflammatory cell infiltration into the retina. A Schematic outlining the PLX5622 (PLX) treatment andexperimental design in this study. Mice were fed with PLX or vehicle control chow for 7 days before the IR injury. B Flow cytometric analysis demonstrated
a significant reduction of Ly6G− CD45+ CD11b+ myeloid cells with PLX treatment at 3 days after the IR in both NIR eyes and IR eyes. C qPCR demonstrated
reductions of chemokine and adhesion molecule expression in the PLX-treated retinas at 16 h after IR. D Leukocytes were labeled with Concanavalin A(ConA) in red. Inhibition of leukocyte adhesion (white arrowheads) in PLX-treated retina 3 days after IR. Data are shown as mean ± SEM, n = 4–9 mice pergroup, where each dot represents an individual eye. *p < 0.05, **p < 0.01, *** p < 0.001 using two-way ANOVA

Zhou et al. Journal of Neuroinflammation(2024) 21:210

finding might indicate that cell types other than myeloid
cells, such as endothelial cells and neurons, could also
express Tnf under the IR condition. PLX treatment did
not affect Vegfa expression in the retina. Consistent witha previous report [36], we did not observe any changes in
Vegfa mRNA expression induced by IR. However, we didobserve increased expression of Angtp2, the gene coding
for Angiopoietin 2, an important regulator of the BRB[44, 45]. Depletion of myeloid cells with PLX significantly
reduced the expression of Angpt2. Claudin-5 protein
is a key protein that regulates the endothelial cell tightjunction. We found that Cldn5 gene expression was notchanged 16 h after IR. However, PLX treatment increased
expression of Cldn5 under the IR condition. This finding
suggests the possibility that myeloid cell depletion could
directly regulate the endothelial cell tight junction andthe blood-retinal barrier function (Fig. 3).
Depleting myeloid cells with PLX5622 ameliorated bloodretinal barrier breakdown in IR

Based on the expression change of inflammatory cytokines as well as Angpt2 and Cldn5 with PLX treatment,
we further investigated whether PLX affected inner
blood-retinal barrier (iBRB) permeability in IR. We firstevaluated retinal vascular permeability change withimmunofluorescence (IF) staining of albumin on retinal

Page 8 of 16

cryosections. Under physiological conditions, albumin
is localized within blood vessels in the retina [46, 47].
As shown in Fig. 4A-B, albumin (green) colocalized with
the CD31+ vessels (yellow) in both CN- and PLX-treatedeyes under the NIR condition, which suggested that the
depletion of myeloid cells under normal conditions did
not affect retinal vascular permeability. At 16 h after IR,
there was a notable increase in albumin extravasation
outside the retinal vessels (white arrowheads) in the CNtreated group (Fig. 4A, right panel). There was a marked
reduction in albumin leakage in PLX-treated retinas
compared to the CN-treated group. Furthermore, evenat 3 days post-IR, the PLX-treated retinas continued to
exhibit a notably smaller area of leakage as depicted
in Fig. 4B (right panel). IBA1 staining (red) also indicated the depletion of myeloid cells with PLX treatment(Fig. 4A, Fig. S2) .
To further confirm our findings from albumin immunofluorescence staining, we quantified the BRB breakdownby measuring the extent of leakage of H3 labeled-mannitol into the retina 3 days after IR. As shown in Fig. 4C,
we did not observe any difference between the PLXand CN-treated eyes under NIR conditions. Consistent
with the immunofluorescence staining results for albumin, a significant reduction of H3-mannitol leakage wasobserved in retinas of PLX-treated eyes at 3 days after IR.

Fig. 3 Myeloid cell depletion regulated inflammatory gene expression in IR. qPCR analysis from the total retina demonstrated that IR induced expressionof multiple inflammatory genes, including Il1b, Il6, Ptgs2, Sphk1, and Tnf. PLX treatment significantly reduced the expression of Il1b, Il6, Ptgs2, and Sphk1,
but not Tnf. PLX treatment also increased the expression of Cldn5, a major endothelial tight junction protein, and Angpt2, an important regulator of endothelial cell permeability. Neither IR nor PLX treatment affected Vegfa expression. Data are shown as mean ± SEM, n = 5–12 mice per group, where each
dot represents an individual eye. *p < 0.05, **p < 0.01, **** p < 0.0001, ns: p > 0.05 using two-way ANOVA

Zhou et al. Journal of Neuroinflammation(2024) 21:210

Page 9 of 16

PLX might affect the ERG function before IR injury. As
shown in Figs. 5A and B and 7 days of PLX treatment did
not affect the a-wave or b-wave of the ERG. We foundthat IR significantly reduced the ERG b-wave amplitude,
but not the a-wave (Fig. 5D). Treatment with PLX did notaffect either the ERG a-wave or b-wave under the IR condition. This finding indicates that depletion of myeloid
cells did not overtly affect neuroretinal function or neuronal degeneration in IR. It has been reported that longterm depletion of myeloid cells with PLX can adversely
affect retinal neuronal function assessed by ERG [49].
The lack of observed effect in our study could be due tothe shorter duration of depletion.
Absence of myeloid cells ameliorated inflammatory gene
expression of Müller glia in IR

Fig. 4 Myeloid cell depletion ameliorated inner blood-retinal barrier
breakdown in IR. A and B Immunofluorescence staining of retinal cryosections demonstrated that there was reduced albumin (green) leakage
outside the CD31+ vessel (yellow) in the PLX-treated retina at 16 h (A) and3 days (B) after IR. White arrowheads indicate albumin extravasated fromblood vessels; C PLX treatment did not affect inner blood-retinal barrier
permeability in the NIR eyes examined by the leakage of H3-mannitol in
the retina after systemic administration. D Depletion of myeloid cells with
PLX significantly reduced inner blood-retinal barrier breakdown at 3 days
after IR. Data are shown as mean ± SEM, n = 10–13 mice per group, where
each dot represents an individual eye. *p < 0.05, ns: p > 0.05 using Student’s
t test

These findings suggest that the depletion of myeloid cells
ameliorated the inner BRB breakdown induced by IR.
Myeloid cell depletion did not affect neuronaldegeneration in IR

We further evaluated whether the depletion of myeloid
cells affects neuronal function in IR using electroretinography (ERG). It has been reported that myeloid cell
depletion with PLX might reduce the retinal function asassessed by ERG in some contexts [48]. We tested thebaseline ERG before IR to exclude the possibility thatWe next investigated the effect of myeloid cell depletionon Müller cells. Müller cells surround retinal blood vessels [8]. Under disease conditions, Müller cells secrete
VEGF and other inflammatory cytokines, which can leadto the breakdown of the iBRB. The cross-talk between
Müller cells and myeloid cells in neurodegeneration has
gained increasing attention in recent years. The role ofinteraction between myeloid cells and Müller cells in regulating the inner BRB function is less clear.
To explore whether depletion of myeloid cells impacts
the iBRB under IR conditions through interactions with
Müller cells, we generated GlastCreER; Rpl22 mice
(Fig. 6A). GlastCreER mice express CreER under the control of the Glast (glial high affinity glutamate transporter)
promoter. When crossed with the Rpl22 mice, it enables
analysis of the translatome in Müller cells. We first confirmed the enrichment of Müller cell-specific RNAs in
the HA-pulldown samples by examining Glast expression (Fig. 6B). We then investigated the expression of
inflammatory factors in Müller cells with PLX treatment.
Expression of many inflammatory factors such as Il1b
and Ptgs2 was barely detectable under NIR conditions
and strongly upregulated in IR in the Müller enrichedsamples (Fig. S3). Under IR conditions, we found that
PLX treatment significantly reduced the expression of
inflammatory genes, including Tnf, Il1b, C3, and Ptgs2(Fig. 6C). We did not observe any change in the Vegf
expression. These data suggest that depleting myeloid
cells ameliorated the expression of inflammatory genes in
Müller cells induced by IR.
To further confirm the direct effect of myeloid cells
on Müller cells, we performed in vitro experiments withmicroglia and Müller cell co-culture (Fig. 6D). After coculturing for 48 h, Müller cells were collected and analyzed using qPCR. We found that co-culturing of Müller
cells with BV2 microglia leads to an increase in theexpression of Tnf. When co-cultured with LPS-activated
BV2 cells, there was a significant upregulation of C3, Il1b,

Zhou et al. Journal of Neuroinflammation(2024) 21:210

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Fig. 5 Myeloid cell depletion did not affect neuronal dysfunction 7 days after IR. A Representative ERG tracings with flash intensity of 7.9 cd*s/m2 fromthe vehicle- or PLX-treated mouse retina after 7 days of treatment. 7-day treatment with PLX did not affect baseline a-wave and b-wave compared to
the vehicle-treated group. B Representative ERG tracings from IR and NIR mice with vehicle or PLX treatment at 7 days after IR. There was no difference
between PLX-treated and vehicle eyes on ERG a-wave and b-wave. Data are shown as mean ± SEM, n = 10–13 mice per group

Ptgs2, and Il6 in the Müller cells (Fig. 6E). These findings are consistent with the in vivo Müller cell RiboTag
experiment and suggest that activated myeloid cells canregulate the expression of inflammatory factors in Müller cells, contributing to and further accentuating iBRB
breakdown in the retina.
We further investigated whether myeloid cells affectendothelial cell barrier function directly or indirectly(through regulation of Müller cells) using an in vitro
endothelial cell assay of diffusive solute flux [33]. We
found that culture medium collected from LPS-treated
BV2 significantly reduced barrier function of cultured
human umbilical vein endothelial cells (HUVEC) compared to culture medium collected from BV2 without LPS (Fig. S3). This finding indicates that activatedmicroglia can directly impair endothelial cell barrier
function. We next investigated whether microglia canregulate EC barrier function indirectly via Müller cells.
We first cultured Müller cells under three conditions – inthe absence of BV2 microglia (“lone” Müller cells), in coculture with untreated BV2 microglia (“quiescent” Müller
cells), and in co-culture with LPS-treated BV2 (“activated” Müller cells). We then removed the microglia andco-cultured the differentially treated Müller cells with
HUVECS (Fig. 6F-G). Interestingly, EC barrier function
was enhanced when co-cultured with “lone” Müller cells.
In addition, EC barrier function was further strengthened in co-culture with “quiescent” Müller cells that had
been exposed to quiescent microglia as compared to“lone” Müller cells. This suggests that quiescent microglia might influence Müller cells toward a phenotype thatenhances EC barrier function. Strikingly, this beneficial
effect on EC barrier function was lost when ECs were
co-cultured with “activated” Müller cells that had been
exposed to LPS-activated microglia. Together, these findings indicate that microglia can regulate vascular cellbarrier function both directly and indirectly through theregulation of Müller cells.

Discussion
Breakdown of the inner blood-retinal barrier is known
to be the critical instigator for macular edema, themost common cause of visual loss in ischemic retinopathies. Intensive research interest has emerged in gaining
insights into the regulation of retinal vascular endothelial integrity by other cellular elements of the retina. In
this current study, we found that the depletion of myeloid
cells with the CSF1R inhibitor PLX5622 (PLX) ameliorated the inner blood-retinal barrier breakdown inducedby ischemia-reperfusion. This finding indicates thatmicroglia/macrophages are important in regulating theinner BRB under disease conditions, highlighting myeloid
cell modulation as a promising therapeutic strategy.
Pharmacologic targeting of CSF1R has been a valuable investigational approach for studying the role ofmicroglia, including in the brain and eye. The CSF1R
inhibitor PLX has been especially useful, based on its
high selectivity and ability to penetrate the blood-retinal

Zhou et al. Journal of Neuroinflammation(2024) 21:210

Page 11 of 16

Fig. 6 Myeloid cells regulate Müller glia activation in IR. A Schematic describing the experimental design of GlastCreER Müller specific-RiboTag mice.
B Enrichment of Müller cell translatome was confirmed with the qPCR analysis of Glast (glial high affinity glutamate transporter) gene expression with
Input and HA-enriched samples. C qPCR analysis showed that PLX treatment significantly reduced the expression of Tnf, C3, Il1b, and Ptgs2 genes on
Müller cells after IR. PLX treatment did not affect the Müller cell Vegf expression. D Experimental design of BV2 and Müller cell culture. E qPCR analysis
demonstrated increased gene expression of Tnf, C3, Il1b, Ptgs2, and Il6 in Müller cells when co-cultured with BV2 cells. F Experimental design of BV2, Müller
cell, and HUVEC co-culture. G HUVECs co-cultured with Müller cells (“lone” Müller cells), or with BV2 previously cocultured with Müller cells (“quiescent”
Müller cells), exhibited reduced diffusive solute flux. HUVECs co-cultured with Müller cells previously co-cultured with LPS-treated BV2 (“activated” Müller
cells) exhibited increased diffusive solute flux. Data are shown as mean ± SEM. For Fig. 6B and C, n = 3–4 mice per group, where each dot represents an
individual eye. *p < 0.05, ns: p > 0.05 using Student’s t test; For Fig. 6E and G, n = 3–4 per group, where each dot represents an individual sample. *p < 0.05,**p < 0.01, *** p < 0.001, **** p < 0.0001, ns: p > 0.05 using one-way ANOVA. ND: non-detectableand blood-brain barriers [15]. It accomplishes highly
efficient depletion of retinal microglia, achieving 98%
depletion after 1 week of treatment [50]. In the current
study, we found that 7-day treatment with PLX markedly reduced the number of CD11b+ and CD45+ cells in

the retina in both non-IR and IR eyes. This indicates a
striking reduction of both retinal microglia and infiltrating monocyte-derived macrophages from the blood following ischemia-reperfusion injury. Notably, our qPCR
analysis at 16 h after IR injury demonstrated that PLX

Zhou et al. Journal of Neuroinflammation(2024) 21:210

treatment resulted in marked downregulation of multiple genes involved in recruitment of inflammatory cells
from the circulation, including the adhesion molecule
ICAM-1 and chemokines such as CCL2. CCL2 is known
to be an important chemokine for macrophage recruitment into the retina during inflammation [51, 52]. Since
microglia are known to secrete and/or regulate thesemolecules [53], our data are consistent with the conceptthat PLX treatment suppresses the reactive microglial
response to IR and with it, a microglial-medicated impetus for recruitment of other inflammatory cells from thecirculation, including monocyte-derived macrophages.
This would be similar to the implication of microglia asa critical initiator of peripheral immune cell infiltrationin an animal model of experimental autoimmune uveitis[19]. It is important to be mindful that PLX treatment,in addition to its effect on depleting resident microglia
in the CNS and retina, can also affect the immune cell
population such as macrophages and dendritic cells inperipheral blood and other tissues/organs such as thespleen, bone marrow, and skin [21]. This effect is especially apparent for longer durations of PLX treatmentcompared to shorter term treatment [18, 21]. With ourshorter-term regimen of PLX treatment, we did notobserve a significant effect on monocyte numbers inthe spleen. Nevertheless, we cannot complete exclude
the possibility that a reduction in systemic, circulating
monocytes could be a contributing factor to the reduction in infiltrating mononuclear phagocytes following IR
with PLX treatment.
The retinal ischemia-reperfusion model directly
impairs the retinal vascular circulation and has therefore
yielded many important insights relating to this vascularbed. Inner BRB breakdown has been well-characterized
in this model [36]. With its primary effect in suppressing
microglia as well as modulating macrophages, we used
PLX to study and implicate the role of these cell populations. We observed that depleting myeloid cells with
the CSF1R inhibitor PLX ameliorated the inner BRB
breakdown, using two independent methods for evaluating retinal vascular leakage. We found that the lack ofmyeloid cells ameliorated the BRB breakdown, suggesting that immune cells are an important factor inducing
the iBRB breakdown. In contrast, we did not observe any
neuronal protection effect from myeloid cell depletionin this IR setting, suggesting a specific effect on vascularregulation. Our study could indicate a primary effect of
microglia, or alternatively a combined effect of microglia and macrophages. This is consistent with previous
studies of microglia/macrophage modulation. Agentsthat are known to modulate microglia and macrophages,
including minocycline and the GLP-1R agonist exendin-4, inhibited inner blood-retinal barrier breakdown
after retinal ischemia-reperfusion [25, 54]. Interestingly,

Page 12 of 16

minocycline treatment suppressed neuroinflammationand had a selective effect in protecting against retinal vascular permeability, without impacting neurodegeneration[25], similar to our own findings. In the STZ-induced diabetic rodent model, depletion of microglia (and possibly
macrophages) alleviated neuronal and vascular damage
in the STZ-induced diabetes model [55]. AAV-mediateddelivery of the anti-inflammatory chemokine fractalkine
in the STZ-diabetes model, reduced fibrinogen leakage
from retinal vessels. This was associated with regulationof microglia-mediated neuroinflammation [56, 57].
Our study indicates the importance of myeloid cells,
also referred to as mononuclear phagocytes, in the breakdown of the inner blood-retinal barrier, in the context
of ischemia-associated injury. Notably, PLX-mediateddepletion was previously investigated in a study of outerblood-retinal barrier breakdown in a mouse model ofendotoxin (LPS)-induced uveitis (EIU) [22]. In this study,
mice were subjected to repeated, systemic challenge withlipopolysaccharide (LPS). As noted by the authors, theprimary BRB defect in these mice was at the level of theretinal pigment epithelium (RPE), the site of the outerblood-retinal barrier. This was manifested by the marked
increase in subretinal fluid in these LPS-challenged mice.
This was associated by fluorescein angiographic studies that showed focal, non-vascular leakage accounting for the subretinal fluid. The authors also observed
an increase in retinal vascular permeability that theyfound to be transient in this model. The study reported
that PLX treatment, with its modulation of mononuclear
phagocytes, suppressed the outer blood-retinal barrier
breakdown. LPS is known to be a strong, direct activatorof microglia and macrophages [58, 59], which could be an
important mechanism for the oBRB dysregulation. This
provides important evidence supporting the ability of themononuclear phagocytes to regulate RPE-mediated barrier function.
A critical concept in the understanding of retinal vascular disease is the pathogenic role of dysfunction of theneurovascular unit, comprising neurons and glial elements in addition to vascular cells [8]. Microglia play animportant role in regulating these cellular elements bothin the retina and brain. In neurodegenerative diseasesof the brain, there is increasing awareness of the importance of crosstalk between microglia and astrocytes,
including the role of microglia in initiating and modulating astrocytic responses to stressors [60–62]. Strikingly,
although retinal microglia are increasingly appreciatedto play significant regulatory roles in multiple retinal
diseases [53], its crosstalk with glial elements promoting
disease progression remains largely undefined, including in the setting of retinal ischemia-reperfusion [7, 34].
In the retina, Müller cells are the predominant glial element, representing 90% of the glia. Müller cell reactivity

Zhou et al. Journal of Neuroinflammation(2024) 21:210

occurs in retinal disease settings, and these reactive
Müller glia can exert both neuroprotective and neurotoxic effects [63, 64]. Müller cells are known to be an
important cell type that contributes to the inflammatoryenvironment in ischemic retinopathies, especially withrespect to their production of cytokines. Müller cells
envelope retinal blood vessels [8], facilitating their regulation of the vasculature. Müller cells have been implicated as an important regulator of neuroinflammation indiabetic retinopathy and vascular leakage, in large partthrough its regulation of the growth factors and cytokine environment. Interestingly, Müller cells can haveboth pathogenic [65, 66] and protective [67] effects in theretina, including vascular barrier function, depending on
the context and the activation state the Müller cells. In
order to study the effect of microglia depletion on Müller cell state, we used a Ribotag approach that allowed us
to analyze cell-specific gene expression in vivo [29, 68].
For Müller cell-specific expression, we used Glast-CreER
transgenic mice, which allows for Müller cell-specificexpression [27]. Strikingly, we found that PLX treatment significantly curtailed Müller cell expression of Tnf,
Il1b, C3, and Ptgs2, which encodes COX-2. In contrast,
there was no effect on Vegfa expression. This indicatesthat reactive microglia and macrophages can provoke an
inflammatory response in Müller cells in retinal IR. As an
additional study, we used an in vitro co-culture system of
microglia and Müller cells. This demonstrated that activated microglia similarly upregulated Müller cell expression of multiple pro-inflammatory genes compared toquiescent microglia. Notably, Müller cell expression of
Tnf was significantly upregulated by both quiescent andactivated microglia in this in vitro system. We also investigated the effect of culture microglia and Müller cells
in regulating endothelial cell barrier function. Interestingly, we found that when co-cultured with unperturbed
Müller cells, the barrier function of endothelial cells wasstrengthened. This barrier function was further enhanced
when the endothelial cells were co-cultured with Müller
cells which had been previously co-cultured with quiescent BV2 microglia. These findings suggest that Müller
cells and microglia can be beneficial in supporting thebarrier function of endothelial cells. However, this beneficial effect on EC barrier function was lost when ECs
were co-cultured with “activated” Müller cells that had
been exposed to LPS-activated microglia. This supports
the ability of reactive microglia to directly activate Müller
cells toward a pathogenic state, further impairing endothelial cell barrier function.
It should be noted that reactive microglia likely regulate other cell types in the context of retinal IR, forinstance vascular cells. For instance, we observed that
PLX treatment resulted in an upregulation of Cldn5 in
IR. This endothelial cell-specific gene encodes the tight

Page 13 of 16

junction protein CLDN5, an important constituent of theiBRB [4].This could be due to the suppression of inflammatory cytokines by myeloid cell depletion, including
TNF-a and IL-1b which are known to induce expressionof Cldn5 [69, 70]. We also observed that the expressionof angiopoietin 2 was significantly reduced with myeloid
cell depletion under the IR condition. Ang2, expressed bymultiple retinal cell types including vascular endothelial
cells, mural cells, and neurons, plays an important role indirectly regulating EC barrier function in the retina.
It is also worthwhile to consider the potential involvement of other cell types in the neurovascular unit. With
respect to macroglia, we focused on Müller cells, since
they are the predominant glial element of the retina
and are well-known to regulate the inner blood-retinal
barrier [71]). Astrocytes have received much less attention for their role in iBRB regulation, although they are
known to play an essential role in developmental retinal
angiogenesis of the superficial retinal vascular plexus[72]. The retinal vessels of the superficial retinal plexus
are surrounded by the specialized feet of Müller cells andastrocytes, while vessels of the intermediate and deep
plexi are only surrounded by Müller cells [71]. Therefore,
it will be of interest to consider the potential involvementof astrocytes in future studies.
Finally, we should note the emerging research on perivascular macrophages as additional resident mononuclear phagocytic cell type in the retina. It has beensuggested that these cells play a role in the maintenanceof the blood-retinal barrier [73]. In addition, their role
in retinal disease pathology is increasingly appreciated,
including the promotion of inflammation in model ofblue light-induced photoreceptor degeneration [74].
Therefore, it will be of great interest in future studies
to investigate the role of perivascular macrophages as
an additional important myeloid cell type involved inregulating the inner blood-retinal barrier. Overall, ourstudy supports a central role for microglia and possibly
monocyte-derived macrophages as mediators of inner
blood-retinal barrier breakdown, indicating an important cellular target of therapeutic modulation of macularedema in a variety of ischemic retinal conditions.

Conclusion
The present study demonstrates the importance ofmononuclear phagocytes as mediators of inner bloodretinal barrier breakdown in the mouse model of retinal
ischemia-reperfusion. PLX5622 treatment efficiently
depletes microglia in healthy retinas, with suppressionof the microglia activation response to retinal IR as well
as of infiltration of monocyte-derived macrophages.
This results in constraint of the overall neuroinflammatory response to IR as well as IR-induced retinal vascular
hyperpermeability. An important aspect of the effect isZhou et al. Journal of Neuroinflammation(2024) 21:210

Page 14 of 16

Fig. 7 Schematic of the proposed role and effect of microglia and mononuclear phagocytes in retinal ischemia-reperfusion. Retinal microglia are activated by IR stress. Reactive microglia secrete chemokines, recruiting monocyte-derived macrophages infiltrating from the retinal circulation. Activatedmyeloid cells (microglial and monocyte-derived macrophages) express inflammatory cytokines that promote inner blood-retinal barrier breakdown. In
addition, microglia (and possibly monocyte-derived macrophages) modulate retinal Müller glia, promoting a neuroinflammatory phenotype including
secretion of inflammatory cytokines by this glial population, further exacerbating inner blood-retinal barrier breakdown. Müller cells thereby play animportant pathogenic role in amplifying the initiating effect of microglial activation

highly likely to be the direct regulation of retinal microglia. In addition, it likely also reflects the regulation ofdownstream effects of microglia, including the stimulation of infiltrating monocyte-derived macrophages
from the circulation. An additional important effect ison microglia-induced activation of Müller glia towarda pathogenic state. These pathogenic Müller cells play
an important role in amplifying the initiating effect of
microglial activation. Our proposed conceptualization of
the role of microglia in triggering the neuroinflammatory
response and iBRB breakdown is depicted in Fig. 7.
Abbreviations
BRB Blood	Retinal barrier
iBRB	Inner blood-retinal barrier
oBRB	Outer blood-retinal barrier
BBB	Blood-brain barrier
CSF-1R	Colony-stimulating factor 1 receptor
PLX	PLX5622
IR	Ischemia reperfusion
NIR	Non-ischemia reperfusion
ERG	ElectroretinogramDR	Diabetic retinopathy
CRVO	Central retinal vein occlusion

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03190-9.
Supplementary Fig. 1. Gating strategy for retinal flow cytometric analysis.
Ly6G-positive cells were excluded prior to analysis of CD11b CD45 expression.
Supplementary Fig. 2. Immunofluorescence staining of IBA1 in retinas
collected after 3 days of IR. Myeloid cells were stained with IBA1 in red.
There was a significant increase in IBA1 + cells in the IR Vehicle groupcompared to the NIR group. There were no IBA1 + cells in the PLX-treated
IR group.
Supplementary Fig. 3. Activated BV2 cells impaired endothelial cellbarrier function. BV2 cells were treated with or without LPS (1 µg/ml) for
72 h. Additional wells with BV2 culture medium with/without LPS but not
BV2 served as controls. After 3 days, the culture medium was collected
and applied to Transwell inserts and wells that were seeded with HUVEC.
Diffusive solute flux assays were done after 24 h. LPS alone did not affect permeability of HUVEC. While culture medium from untreated BV2strengthened HUVEC barrier function, culture medium from LPS-treated
BV2 impaired HUVEC barrier function. n = 6 per group, where each dot
represents an individual sample. * **p < 0.01, **** p < 0.0001, ns: p > 0.05
using one-way ANOVA.
Supplementary Fig. 4. Increased expression of Tnf and C3 in Müller cells
3 days after IR. Expression was evaluated using the RiboTag method. No
difference was found in expression of Vegfa. **p < 0.01, ns: p > 0.05 using
Student’s t test.

Zhou et al. Journal of Neuroinflammation(2024) 21:210

Supplementary Fig. 5. PLX treatment reduced macrophage number, but
not monocyte number in the spleen. Spleens were collected after 2 weeks
of PLX treatment and analyzed by flow cytometry. There was a reduction
in number of CD11b+CD45+Ly6G−CD11c−Ly6C− macrophages. No difference was found in CD11b+CD45+Ly6G−CD11c−Ly6C+ monocyte number.
n = 5 *p < 0.05, ns: p > 0.05 using Student’s t test.
Acknowledgements
This study was supported by Plexxikon in its provision of PLX5622. We
recognized the support from JHU Ross Flow Cytometry Core.
Author contributions
LZ, ED, and ZX developed the concept of the study, designed the experiments,
and wrote the manuscript. LZ performed the experiments and data analysis.
ZX and MR performed the cell culture study. HL, HC, YX, and GL performed theanimal experiments.
Funding
This work was supported by research grants from the National Institutes of
Health: EY022683 (to E.J.D.), the Altsheler-Durell Foundation, P30EY001765(toWilmer Imaging and Microscopy Core Module), P30AR070254 (to the Johns
Hopkins Rheumatic Disease Research Core Center), and unrestricted funds
from the Research to Prevent Blindness.
Data availability
The data sets used and analyzed from the current study are available from thecorresponding author upon reasonable request.

Declarations
Ethical approval
Animal studies were approved by the Institutional Animal Care and
Use Committee of the Johns Hopkins University School of Medicine. Allprocedures involving animals were conducted under the Association for
Research in Vision and Ophthalmology Statement for the Use of Animals in
Ophthalmic and Vision Research.
Competing interests
The authors declare no competing interests.
Received: 16 May 2024 / Accepted: 29 July 2024

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