Effect of Inhibition of Colony Stimulating Factor 1 Receptor on Choroidal Neovascularization in Mice.

Neovascular age-related macular degeneration is one of the leading causes of blindness. Microglia and macrophages play critical role in choroidal neovascularization (CNV) and may therefore be potential targets to modulate the disease course. This study evaluated the effect of the colony stimulating factor-1 receptor (CSF-1R) inhibitor PLX5622 on experimental laser-induced CNV. A 98% reduction of retinal microglia cells was observed in the retina one week after initiation of PLX5622 treatment, preventing accumulation of macrophages within the laser site and leading to a reduction of leukocytes within the choroid after CNV induction. Mice treated with PLX5622 had a significantly faster decrease of the CNV lesion size as revealed by in vivo imaging and immunohistochemistry from day 3 to day 14 compared to untreated mice. Several inflammatory modulators, such as CCL9, granulocyte-macrophage colony-stimulating factor, ssoluble tumor necrosis factor receptor-I, interleukin-1α, and matrix metallopeptidase-2 were elevated in the acute phase of the disease when microglia were ablated with PLX5622, whereas other cytokines (eg, interferon-γ, interleukin-4, and interleukin-10) were reduced. Our results suggest that CSF-1R inhibition may be a novel therapeutic target in patients with neovascular age-related macular degeneration

Multimodal imaging of experimental choroidal neovascularization.

AIM To compare choroidal neovascularization (CNV) lesion measurements obtained by in vivo imaging modalities, with whole mount histological preparations stained with isolectin GS-IB4, using a murine laser-induced CNV model. METHODS B6N.Cg-Tg(Csf1r-EGFP)1Hume/J heterozygous adult mice were subjected to laser-induced CNV and were monitored by fluorescein angiography (FA), multicolor (MC) fundus imaging and optical coherence tomography angiography (OCTA) at day 14 after CNV induction. Choroidal-retinal pigment epithelium (RPE) whole mounts were prepared at the end of the experiment and were stained with isolectin GS-IB4. CNV areas were measured in all different imaging modalities at day 14 after CNV from three independent raters and were compared to choroidal-RPE whole mounts. Intraclass correlation coefficient (ICC) type 2 (2-way random model) and its 95% confidence intervals (CI) were calculated to measure the correlation between different raters' measurements. Spearman's rank correlation coefficient (Spearman's r) was calculated for the comparison between FA, MC and OCTA data and histology data. RESULTS FA (early and late) and MC correlates well with the CNV measurements ex vivo with FA having slightly better correlation than MC (FA early Spearman's r=0.7642, FA late Spearman's r=0.7097, and MC Spearman's r=0.7418), while the interobserver reliability was good for both techniques (FA early ICC=0.976, FA late ICC=0.964, and MC ICC=0.846). In contrast, OCTA showed a poor correlation with ex vivo measurements (Spearman's r=0.05716) and high variability between different raters (ICC=0.603). CONCLUSION This study suggests that FA and MC imaging could be used for the evaluation of CNV areas in vivo while caution must be taken and comparison studies should be performed when OCTA is employed as a CNV monitoring tool in small rodents

Dramatic Effect of Oral CSF-1R Kinase Inhibitor on Retinal Microglia Revealed by In Vivo Scanning Laser Ophthalmoscopy

This report provides sound evidence that the small molecule pharmaceutical PLX5622, a highly selective CSF-1R kinase inhibitor, crosses the blood-retina barrier and suppresses microglia activity. Members of this class of drug are in advanced clinical development stages and may represent a novel approach to modulate ocular inflammatory processes

Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure.

BACKGROUND Clustering of microglia around the vasculature has been reported in the retina and the brain after systemic administration of lipopolysaccharides (LPS) in mice. LPS acts via activation of Toll-like receptor 4 (TRL4), which is expressed in several cell types including microglia, monocytes and vascular endothelial cells. The purpose of this study was to investigate the effect of systemic LPS in the pigmented mouse retina and the involvement of endothelial TLR4 in LPS-induced retinal microglia activation. METHODS C57BL/6J, conditional knockout mice that lack Tlr4 expression selectively on endothelial cells (TekCre-posTlr4loxP/loxP) and TekCre-negTlr4loxP/loxP mice were used. The mice were injected with 1 mg/kg LPS via the tail vein once per day for a total of 4 days. Prior to initiation of LPS injections and approximately 5 h after the last injection, in vivo imaging using fluorescein angiography and spectral-domain optical coherence tomography was performed. Immunohistochemistry, flow cytometry, electroretinography and transmission electron microscopy were utilized to investigate the role of endothelial TLR4 in LPS-induced microglia activation and retinal function. RESULTS Activation of microglia, infiltration of monocyte-derived macrophages, impaired ribbon synapse organization and retinal dysfunction were observed after the LPS exposure in C57BL/6J and TekCre-negTlr4loxP/loxP mice. None of these effects were observed in the retinas of conditional Tlr4 knockout mice after the LPS challenge. CONCLUSIONS The findings of the present study suggest that systemic LPS exposure can have detrimental effects in the healthy retina and that TLR4 expressed on endothelial cells is essential for retinal microglia activation and retinal dysfunction upon systemic LPS challenge. This important finding provides new insights into the role of microglia-endothelial cell interaction in inflammatory retinal disease

Systemic Lipopolysaccharide Exposure Exacerbates Choroidal Neovascularization in Mice.

This study aims to investigate the effect of a systemic lipopolysaccharide (LPS) stimulus in the course of laser-induced choroidal neovascularization (CNV) in C57BL/6 J mice. A group of CNV-subjected mice received 1 mg/kg LPS via the tail vein immediately after CNV induction. Mouse eyes were monitored in vivo with fluorescein angiography for 2 weeks. In situ hybridization and flow cytometry were performed in the retina at different time points. LPS led to increased fluorescein leakage 3 days after CNV, correlated with a large influx of monocyte-derived macrophages and increase of pro-inflammatory microglia/macrophages in the retina. Additionally, LPS enhanced Vegfα mRNA expression by Glul-expressing cells but not Aif1 positive microglia/macrophages in the laser lesion. These findings suggest that systemic LPS exposure has transient detrimental effects in the course of CNV through activation of microglia/macrophages to a pro-inflammatory phenotype and supports the important role of these cells in the CNV course

Semi-quantitative analysis of cytokine and chemokine levels in the eyes of CNV-subjected C57BL/6J mice.

Mice fed with normal mouse chow or chow containing the colony-stimulating factor-1 receptor (CSF-1R) inhibitor PLX5622 for one week and until the end of the experiments, were subjected to laser-induced choroidal neovascularization (CNV). The posterior part of the mouse eyes was isolated 3 and 7 days after the laser, total protein were extracted and antibody array membranes were employed to analyze cytokine levels during the course of CNV in the presence or absence of PLX5622. The data were analyzed using the “Protein Array Analyzer” function of the ImageJ software and the signal of each individual spot was normalized between different membranes using the positive control spots

Study of the neuroprotective action of cannabinoids in retinal ischemia

Retinal ischemia leads to neovascularization and excitotoxicity (cell death) both of which can lead to the development of very severe retinopathies, such as diabetic retinopathy and age related macular degeneration (Lipton and Rosenberg, 1994; Campochiaro, 2000; Osborne et al., 2004; Grant et al., 2005), that often result in poor visual acuity and blindness. Surgical and new pharmacological approaches are available to target the neovascular component of ischemia induced retinopathies but not the neurodegenerative component. In order to preserve vision both components must be treated. Therefore, it is mandatory to establish new pharmacological targets for the treatment of the neurodegenerative component. The main aim of the present study was to investigate whether the endocannabinoid system may serve as an efficacious therapeutic target for the treatment of neurodegenerative retinal disease. Specifically, the neuroprotective actions of the endocannabinoids AEA (N-arachidonoylethanolamine, anandamide) and 2-AG (2-arachidonoylglycerol) and synthetic cannabinoids MethAEA (Methanandamide) and HU-210 were studied, as well as the neuroprotective actions of the inhibitors of the enzymes responsible for the metabolism of the endocannabinoids. In addition, the involvement of the cannabinoid CB1 and CB2 receptors and the TRPV1 vallinoid receptor in the neuroprotective actions of endo- and synthetic cannabinoids, as well as the downstream signaling pathways leading to their putative neuroprotective effects were also investigated. We employed the in vivo AMPA model of retinal excitotoxicity, previously developed in our laboratory (Kiagiadaki and Thermos, 2008) and we investigated the neuroprotective actions of the synthetic HU-210 and MethAEA and the endogenous cannabinoids AEA and 2-AG. Cannabinoids were intravitreally injected with AMPA (42nmoles/eye) and 24 hours post injection, the animals were euthanized and their retinas were prepared for immunohistochemical studies or western blot analysis.AMPA caused a reduction of bNOS (brain nitric oxide synthase) and ChAT (choline acetyltransferase) immunoreactive neurons compared to the control tissues and the cannabinoids afforded neuroprotection against AMPA excitotoxicity, increasing the number of bNOS and ChAT immunoreactive cells compared to the AMPA treated tissues. The toxic effects of AMPA and the neuroprotective actions of cannabinoids were also substantiated by the TUNEL assay. Additional studies revealed that the cell death observed in the presence of AMPA does not involve activation of the apoptosis mediator caspase-3. To investigate the involvement of CB1 and CB2 receptors in the neuroprotection, we performed a series of studies, namely the intravitreal co-injection of cannabinoid receptor antagonists with AMPA and cannabinoids, radioligand binding studies, study of the neuroprotective actions of the CB2 selective agonist JWH015, RT-PCR and studies in CB1-/-andCB2-/- mice, which revealed that the CB1 but not the CB2 receptor is involved in the neuroprotective actions of cannabinoids. We also performed pharmacological studies to assess the involvement of the TRPV1 vallinoid receptor in the cannabinoids’ neuroprotective actions. The data obtained from these studies revealed that the TRPV1 receptor, is not involved in the neuroprotective actions of cannabinoids in the vivo retinal model of AMPA excitotoxicity. Having assessed the neuroprotective properties of the cannabinoids in the AMPA model of excitotoxicity in the retina and the involvement of the CB1 cannabinoid receptor in their actions, we subsequently investigated the signaling pathways that lead to the neuroprotection. We performed western blot analysis against phosphorylated and total forms of the PI3K/Akt and MEK/ERK1/2 kinases, functional studies using the PI3K/Akt inhibitor wortmannin and neuroprotection studies in Akt2-/- mice. The data obtained suggested the involvement of PI3K/Akt and/or MEK/ERK1/2 signaling pathways in the neuroprotection afforded by the cannabinoids. Specifically, the PI3K/Akt signaling pathway seems to be involved in the neuroprotective actions of ΑΕΑ, 2-AG and HU-210, while the MEK/ERK1/2 pathway is involved in the neuroprotective actions of ΑΕΑ and 2-AG, but not those of HU-210. Besides the direct activation of cannabinoid receptors with specific agonists, cannabinergic signaling can also be modulated through inhibition of endocannabinoid metabolism. Therefore, we examined the putative neuroprotective actions of the inhibitors of the two metabolic enzymes of AEA and 2-AG (Fatty Acid Amide Hydrolase, FAAH and Monoacylglycerol lipase, MGL for AEA and 2-AG, respectively), to substantiate whether these inhibitors could provide a new therapeutic target for the treatment of retinal disease. We performed experiments employing a FAAH inhibitor (AM6642), which inhibits the degradation of AEA and a dual FAAH/MGL inhibitor (AM9928), which inhibits the degradation of both AEA and 2-AG. These compounds afforded neuroprotection when co-injected with AMPA, with AM9928 affording greater neuroprotection than AM6642.The neuroprotective actions of the two inhibitors were reversed in the presence of the CB1 receptor antagonist AM251. Subsequently, we co-injected the above mentioned enzyme inhibitors with AMPA plus endogenous and synthetic cannabinoids to examine if the neuroprotective effects could be increased due to the elevation of endocannabinoid levels in the synapse and the exogenously added agents. Both the FAAH and the FAAH/MGL inhibitors failed to afford neuroprotection in the presence of exogenously applied cannabinoids. These results suggest that blockade of endocannabinoids’ degradation protects the retina from the AMPA insults. However, the co-administration of the enzyme inhibitors and exogenously administered endocannabinoids did not have an additive effect, probably due to desensitization and downregulation of CB1 receptor (Schlosburget al., 2010). This tenet must be investigated further. MethAEA, a metabolically stable analogue of AEA that is metabolized by pathways independent of FAAH, did not alter the neuroprotective action of AM6642 (Abadji et al., 1994). The second synthetic cannabinoid employed (HU-210) reversed the neuroprotective actions of both AM6642 and AM9928. Since there are no reports in the literature regarding the metabolic pathways of HU-210, one cannot make any hypotheses regarding HU-210’s actions.The data obtained from the present study suggest that endogenous and synthetic cannabinoids protect the retina from AMPA excitotoxicity, via activation of the CB1, but not CB2 or TRPV1 receptors, with the involvement of the PI3K/Akt and/or MEK/ERK1/2 signaling pathways. Inhibition of the enzymes that metabolize the endocannabinoids also afforded neuroprotection, yet the co-administration of exogenous endocannabinoids leads to its attenuation. In conclusion, the present study revealed an important role for the endocannabinoid system in the physiology and pathophysiology of the retina. This system could provide an important therapeutic target for the development of more efficacious therapeutics for the treatment of retinopathies whose pathophysiology involves excitotoxic insults.Η διεγερσιτοξικότητα είναι μια άμεση συνέπεια της αμφιβληστροειδικής ισχαιμίας η οποία μπορεί να οδηγήσει στην ανάπτυξη σοβαρών αμφιβληστροειδοπαθειών όπως είναι η διαβητική αμφιβληστροειδοπάθεια και η γεροντική εκφύλιση της ωχράς κηλίδας (Lipton και Rosenberg, 1994˙ Campochiaro, 2000˙ Osborne και συν., 2004˙ Grant και συν., 2005). Οι θεραπείες που χρησιμοποιούνται σήμερα για τις ισχαιμικές αμφιβληστροειδοπάθειες περιλαμβάνουν θεραπείες με laser αλλά και φαρμακολογικές προσεγγίσεις που στοχεύουν στην καταπολέμηση της νεοαγγείωσης αλλά όχι και του κυτταρικού θανάτου που παρατηρείται κατά τις ισχαιμικές αμφιβληστροειδοπάθειες. Για τη διατήρηση της όρασης είναι επιτακτική η ανάγκη εύρεσης νέων θεραπευτικών στόχων, που σε συνδυασμό με τις ήδη εφαρμοζόμενες θεραπείες θα μπορούσαν να οδηγήσουν στη δημιουργία πιο αποτελεσματικών θεραπευτικών σχημάτων. Στόχος της παρούσας διατριβής ήταν η διερεύνηση των νευροπροστατευτικών δράσεων των ενδογενών [2-αραχιδονοϋλαιθανολαμίνη (2-arachidonoylethanolamine, ΑΕΑ, anandamide, ανανδαμίδιο) και 2-αραχιδονοϋλγλυκερόλη (2-arachidonoylglycerol, 2-AG)], συνθετικών κανναβινοειδών (MethAEA και HU-210), καθώς και των αναστολέων των ενζύμων μεταβολισμού των ενδοκανναβινοειδών, έναντι του κυτταρικού θανάτου, σε ένα in vivo μοντέλο AMPA διεγερσιτοξικότητας σε αμφιβληστροειδή τρωκτικών, καθώς και των μηχανισμών που εμπλέκονται σε αυτές τις δράσεις. Χρησιμοποιήθηκε το in vivo μοντέλο της AMPA διεγερσιτοξικότητας (Kiagiadaki και Thermos, 2008) στον αμφιβληστροειδή επίμυος και μελετήθηκαν οι νευροπροστατευτικές δράσεις των συνθετικών HU-210 και MethAEA καθώς και των ενδογενών κανναβινοειδών AEA και 2-AG. Τα κανναβινοειδή συν-χορηγήθηκαν ενδοϋαλοειδικά μαζί με AMPA, εικοσιτέσσερις (24) ώρες μετά τη χορήγηση του AMPA τα ζώα θανατώθηκαν και οι αμφιβληστροειδείς τους προετοιμάστηκαν για ανοσοϊστοχημικές μελέτες ή μελέτες ανοσοαποτύπωσης. Το AMPA προκάλεσε ραγδαία μείωση των χολινεργικών και bNOS ανοσοδραστικών κυττάρων συγκριτικά με τους ιστούς ελέγχου, ενώ τα κανναβινοειδή παρείχαν νευροπροστασία έναντι της AMPA διεγερσιτοξικότητας, αυξάνοντας στατιστικώς σημαντικά τoν αριθμό των παραπάνω κυττάρων συγκριτικά με τους ιστούς που έλαβαν AMPA. Οι νευροτοξικές δράσεις του AMPA και οι νευροπροστατευτικές δράσεις των κανναβινοειδών επιβεβαιώθηκαν με τη χρήση της χρώσης TUNEL. Περαιτέρω μελέτες έδειξαν ότι ο κυτταρικός θάνατος που παρατηρήθηκε ήταν ανεξάρτητος της ενεργοποίησης της κασπάσης-3. Για τη μελέτη της εμπλοκής των CB1 και CB2 υποδοχέων στις νευροπροστατευτικές δράσεις των κανναβινοειδών χρησιμοποιήθηκε μια σειρά από διαφορετικές προσεγγίσεις (χρήση ανταγωνιστών των CB1 και CB2 υποδοχέων, μελέτες δέσμευσης, μελέτες της νευροπροστατευτικής δράσης του CB2 αγωνιστή JWH015, μελέτες RT-PCR αλλά και μελέτες σε CB1-/- και CB2-/- μύες), τα αποτελέσματα των οποίων μας οδήγησαν στην υπόθεση ότι ο CB1 αλλά όχι ο CB2 υποδοχέας εμπλέκεται στη νευροπροστασία που παρατηρήθηκε στην παρούσα μελέτη.Μελετήθηκε επίσης η εμπλοκή του TRPV1 υποδοχέα των βανιλλοειδών στις νευροπροστατευτικές δράσεις των κανναβινοειδών με χρήση του ανταγωνιστή (capsazepine) και του αγωνιστή (capsaicin) του TRPV1 υποδοχέα και φάνηκε ότι ο εν λόγω υποδοχέας δεν εμπλέκεται στις δράσεις των κανναβινοειδών που παρατηρούνται στην παρούσα μελέτη. Επόμενο βήμα ήταν η μελέτη της εμπλοκής των σηματοδοτικών μονοπατιών PI3K/Akt και MEK/ERK1/2 στις νευροπροστατευτικές δράσεις των κανναβινοειδών. Μελέτες ανοσοαποτύπωσης καθώς και η χρήση του PI3K/Akt αναστολέα wortmannin αλλά και διαγονιδιακών Akt2-/-μυών υποστήριξαν την εμπλοκή του PI3K/Akt ή/και MEK/ERK1/2 στις νευροπροστατευτικές δράσεις των κανναβινοειδών. Συγκεκριμένα το σηματοδοτικό μονοπάτι PI3K/Akt φαίνεται να εμπλέκεται στις δράσεις των ΑΕΑ, 2-AG και HU-210, ενώ το μονοπάτι MEK/ERK1/2 φαίνεται να εμπλέκεται στις δράσεις των ΑΕΑ, 2-AG, αλλά όχι του HU-210. Πέραν της εξωγενούς χορήγησης των ενδοκανναβινοειδών ΑΕΑ και 2-AG, έμμεσος τρόπος αύξησης των επιπέδων τους στη σύναψη είναι και η αναστολή της αποικοδόμησης τους μέσω αναστολέων των ενζύμων που ευθύνονται για τον καταβολισμό τους [υδρολάση των αμιδίων των λιπαρών οξέων (Fatty Acid Amide Hydrolase, FAAH) και λιπάσης της μονοακύλογλυκερόλης (MonoacylGlycerol Lipase, MGL)], αντίστοιχα. Στην παρούσα μελέτη ερευνήσαμε την πιθανή νευροπροστατευτική δράση του αναστολέα της FAAH (AM6642) και ενός διπλού αναστολέα των FAAH και MGL (AM9928), ως νέους θεραπευτικούς στόχους. Οι δύο αναστολείς συν-χορηγούμενοι με AMPA παρείχαν προστασία στον ιστό με τον ΑΜ9928 να δρα πιο αποτελεσματικά. Η νευροπροστατευτική δράση των δύο αναστολέων αναστάλθηκε παρουσία του CB1 ανταγωνιστή AM251. Για την περαιτέρω αύξηση των επιπέδων των κανναβινοειδών στη σύναψη συν-χορηγήθηκε AMPA με AM6642 ή AM9928 και HU-210, AEA, MethAEA ή 2-AG και μελετήθηκε η ενδεχόμενη νευροπροστατευτική τους δράση. H παρουσία εξωγενώς χορηγούμενων κανναβινοειδών αναίρεσε τη νευροπροστατευτική δράση των AM6642 και AM9928, με μοναδική εξαίρεση τη συν-χορήγηση AMPA + MethAEA + AM6642. Η μείωση που παρατηρήθηκε πιθανόν να οφείλεται σε μειο-ρύθμιση (down regulation) του CB1 υποδοχέα καθώς υπάρχουν δεδομένα που υποστηρίζουν την απευαισθητοποίησή του CB1 και μειορύθμιση του μετά από παρατεταμένη αναστολή του ενζύμου MGL (Schlosburg και συν., 2010). Το γεγονός ότι η MethAEA δε μείωσε τη νευροπροστατευτική δράση του ΑΜ6642 πιθανώς οφείλεται στο ότι η MethAEA είναι ένα μεταβολικά σταθερό μόριο που δεν καταβολίζεται από την FAAH (Abadji και συν., 1994). Το δεύτερο συνθετικό κανναβινοειδές που χρησιμοποιήθηκε (HU-210) συμπεριφέρθηκε μιμούμενο τα ενδογενή κανναβινοειδή και αναίρεσε τις νευροπροστατευτικές δράσεις των ΑΜ6642 και ΑΜ9928. Ωστόσο, δεδομένου ότι δεν υπάρχουν δεδομένα για τον μηχανισμό καταβολισμού του HU-210 δε μπορούμε να σχολιάσουμε τον τρόπο δράσης του HU-210 στη μείωση των νευροπροστατευτικών δράσεων των αναστολέων μεταβολισμού των ενδοκανναβινοειδών. Συμπερασματικά, τα αποτελέσματα της παρούσας διατριβής συνηγορούν υπέρ του νευροπροστατευτικού ρόλου του κανναβινοειδικού συστήματος έναντι της AMPA διεγερσιτοξικότητας στον αμφιβληστροειδή τρωκτικών. Τα κανναβινοειδή παρείχαν προστασία έναντι της AMPA διεγερσιτοξικότητας μέσω ενεργοποίησης του CB1 υποδοχέα και των σηματοδοτικών μονοπατιών PI3K/Akt ή/και ERK1/2. Η μελέτη της εμπλοκής αποπτωτικών μηχανισμών ανέδειξε ότι η ενεργοποίηση της κασπάσης-3 δεν εμπλέκεται στον κυτταρικό θάνατο που παρατηρείται μετά τη χορήγηση AMPA, γεγονός το οποίο απαιτεί περαιτέρω μελέτη. Η αναστολή των ενζύμων καταβολισμού των ενδοκανναβινοειδών φάνηκε εξίσου αποτελεσματική με την εξωγενή χορήγηση κανναβινοειδών στην παροχή νευροπροστασίας στον ιστό. Ωστόσο, παρουσία εξωγενώς χορηγούμενων κανναβινοειδών, οι αναστολείς των ενζύμων καταβολισμού των ενδοκανναβινοειδών δεν παρείχαν νευροπροστατευτική δράση, γεγονός το οποίο χρήζει περαιτέρω διερεύνησης.Τα ευρήματα της παρούσας μελέτης συνηγορούν υπέρ του ότι το κανναβινοειδικό σύστημα μπορεί να διαδραματίσει σημαντικό ρόλο στην φυσιολογία και παθοφυσιολογία του αμφιβληστροειδούς και αποτελεί έναν σημαντικό νευροπροστατευτικό στόχο για τη δημιουργία αποτελεσματικότερων σχημάτων που θα στοχεύουν στη θεραπεία σοβαρών αμφιβληστροειδοπαθειών

Microglia Activation and Recruitment of Circulating Macrophages During Ischemic Experimental Branch Retinal Vein Occlusion

Purpose: To characterize retinal microglia activation and macrophage recruitment in experimental branch retinal vein occlusion (BRVO). Methods: Experimental BRVO was induced in Balb/c mice and histologic changes were studied. Tissue hypoxia was visualized using pimonidazole hydrochloride. Monocyte-derived retinal cells were quantified using histology and flow cytometry. To investigate the dynamics of invading blood-borne macrophages, chimera mice were generated using bone marrow grafts from Cx3cr1(gfp/gfp) mice to rescue lethally irradiated wild-type BALB/c mice. Longitudinal in vivo imaging was performed to monitor cell invasion. The levels of proinflammatory cytokines in the retina were quantified by quantitative real-time PCR. Results: Histology showed disruption of tissue architecture and temporary swelling with marked hypoxia coinciding with increased VEGF-A and hypoxia inducible factor-1α (HIF-1α) expression and elevation of proinflammatory cytokines within 3 days after experimental BRVO, followed by thinning of the inner retinal layers at later time points. Proinflammatory cytokine levels were elevated. Activation of resident retinal microglia and recruitment of circulating macrophages in areas of hypoxic retina were evident early after the insult and peaked at day 7, remaining elevated for up to 28 days. Flow cytometry showed upregulation of CD68 and major histocompatibility complex class-II (MHC-II) expression at day 3, culminating at day 7. Conclusions: Experimental BRVO causes hypoxia and breakdown of the inner blood–retina barrier, followed by activation of microglia and invasion of macrophages from the systemic circulation. Consequently, treatments targeting microglia activation or macrophage recruitment might potentially mitigate the sequelae and attenuate degenerative changes induced by retinal vein occlusion

Colony-stimulating factor 1 receptor inhibition prevents disruption of the blood-retina barrier during chronic inflammation

Abstract Background Microglia-associated inflammation is closely related to the pathogenesis of various retinal diseases such as uveitis and diabetic retinopathy, which are associated with increased vascular permeability. In this study, we investigated the effect of systemic lipopolysaccharide (LPS) exposure to activation and proliferation of retinal microglia /macrophages. Methods Balb/c and Cx3cr1 gfp/+ mice were challenged with LPS (1 mg/kg) daily for four consecutive days. For microglia depletion, mice were treated with colony-stimulating factor 1 receptor (CSF-1R) inhibitor PLX5622 1 week before the first LPS challenge and until the end of the experiment. In vivo imaging of the retina was performed on days 4 and 7 after the first LPS challenge, using optical coherence tomography and fluorescein angiography. Flow cytometry analysis, retinal whole mount, and retinal sections were used to investigate microglia and macrophage infiltration and proliferation after LPS challenge. Cytokines were analyzed in the blood as well as in the retina. Data analysis was performed using unpaired t tests, repeated measures one-way ANOVA, or ordinary one-way ANOVA followed by Tukey’s post hoc analysis. Kruskal-Wallis test followed by Dunn’s multiple comparison tests was used for the analysis of non-normally distributed data. Results Repeated LPS challenge led to activation and proliferation of retinal microglia, infiltration of monocyte-derived macrophages into the retina, and breakdown of the blood-retina barrier (BRB) accompanied by accumulation of sub-retinal fluid. Using in vivo imaging, we show that the breakdown of the BRB is highly reproducible but transitory. Acute but not chronic systemic exposure to LPS triggered a robust release of inflammatory mediators in the retina with minimal effects in the blood plasma. Inhibition of the CSF-1R by PLX5622 resulted in depletion of retinal microglia, suppression of cytokine production in the retina, and prevention of BRB breakdown. Conclusions These findings suggest that microglia/macrophages play an important role in the pathology of retinal disorders characterized by breakdown of the BRB, and suppression of their activation may be a potential therapeutic target for such retinopathies