Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy

Abstract

Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.

Keywords: capillary regulation; diabetes; fractalkine; microglia; retina.

Fig. 1.

Retinal microglia associate with vasculature and neuronal synapses. (A) Whole-mounted mouse retina (Cx3cr1^GFP/+) was labeled with anti-EGFP (microglia, green), and G. simplicifolia IB4 (blood vessels, red). The highlighted region shows microglial association with vessels within the superficial vascular plexus (Inset). (Scale bars, 500 µm; 50 µm, Inset.) (B) The association of microglial processes with vessels of different diameters within the superficial plexus was quantified relative to vessel area for each vessel size and show microglia preferentially associate with capillaries, *P < 0.05, ***P < 0.001. (C) The ultrastructure of microglia–vessel contact within the Cx3cr1^GFP/+ retina shows microglial processes (immunolabeled against EGFP, black dots) adjoin pericytes, which contact the endothelial cells lining the capillary lumen. (Scale bar, 0.5 µm.) (D) A whole-mounted retina from the NG2-DsRed pericyte reporter mouse (pericyte somata, processes, red) stained with Iba-1 (microglia, green) and DAPI (nuclei, blue) shows a microglial process making contact with pericyte somata. The boxed region is shown in orthogonal projections (above and right). (Scale bar, 10 µm.) (E) A high-resolution–rendered image of microglial–pericyte contact taken from asterisk in D. (Scale bar, 5 µm.) (F) Microglial–pericyte interaction was further probed in rat retina and the extent of contact with pericyte somata, processes (NG2⁻) and capillary areas lacking pericyte contact (NG2⁻/IB4⁺) quantified. (G) A vertical section from a Cx3cr1^GFP/+ retina labeled for blood vessels (IB4, magenta), microglia (EGFP, green), neuronal synapses (VGLUT1, red), and cell nuclei (DAPI, blue), showing microglia contact retinal vessels (asterisk) and neuronal synapses (arrowheads). The boxed region was imaged at higher resolution and rendered to highlight microglial–synapse interaction (Inset). (Scale bars, 50 µm; 5 µm, Inset.) (H) Neuronal–microglial–vascular contact is also observed in human retina (microglia, Iba-1, green; vessels, vitronectin, magenta, asterisk; neuronal synapses, VGLUT1, red, arrowheads; cell nuclei, DAPI, blue). (Scale bar, 50 µm.) (I) When neuronal–microglial contact was quantified in the Cx3cr1^+/GFP mouse at the level of the inner retina (vessels, IB4, red; microglia, EGFP, green; neuronal synapses VGLUT1, blue), the majority of microglia contact both neuronal synapses and vessels. (Scale bar, 20 µm.) Data presented as mean ± SEM, n = 5 (B and F), n = 3 (I, Inset). GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; MC, microglia; PC, pericyte; EC, endothelial cell; CL, capillary lumen; ONL, outer nuclear layer; OPL, outer plexiform layer.

Fig. 2.

Microglia constrict retinal capillaries via fractalkine-Cx3cr1 signaling and express genes for vasoactive agents. (A) Ex vivo Cx3cr1^GFP/+ retinae (EGFP; microglia, green) were labeled with Rhodamine B (blood vessels, red) and imaged under live cell microscopy. (Scale bar, 50 μm.) (B) The addition of fractalkine (200 ng/mL) induced vasoconstriction at sites of microglial contact (m+, n = 4 PBS, n = 6 FKN), while no significant vessel alteration occurred in areas lacking microglial processes (m−, n = 5 PBS, n = 6 FKN). When performed on Cx3cr1^GFP/GFP retinae, no constriction was evident (n = 5). Supporting a Cx3cr1-mediated effect, the addition of the small-molecule Cx3cr1 inhibitor, AZD8797, blocked vasoconstriction (Inset, n = 4 FKN, n = 3 FKN+AZD8797). (C) The response of brain vasculature to fractalkine was tested in rat thin scull preparations, with constriction evident 120 s postinjection (n = 3 PBS, FKN). The Insets show representative images at baseline and after fractalkine addition. (Scale bar, 250 μm.) (D) Retinal microglia (EGFP, green), neuronal synapses (VGLUT1, red), and blood vessels (IB4, light blue) were imaged in Cx3cr1^GFP/+ and Cx3cr1^GFP/GFP animals and the extent of vascular and neuronal contact quantified relative to microglial volume (see isolated microglia: red, neuronal contacts; blue, vascular contacts). (Scale bar, 15 μm.) (E) Grouped data showed Cx3cr1^GFP/GFP retinae to have reduced vascular contacts compared to Cx3cr1^GFP/+ retinae (n = 5), while there was no difference in neuronal contacts. Cx3cr1^GFP/GFP microglia exhibited reduced process branching (n = 5). (F) Using in vivo OCTA, retinal capillary diameter was increased in Cx3cr1^GFP/GFP animals compared to Cx3cr1^GFP/+ retinae (n = 4 C57Bl6, n = 6 Cx3cr1^GFP/+, Cx3cr1^GFP/GFP), while there was no alteration in the diameter of arterioles or venules (A/V ratio shown in table, n = 4 C57Bl6, n = 6 Cx3cr1^GFP/+, n = 5 Cx3cr1^GFP/GFP). (G) RNA-seq was performed on FACS-isolated rat retinal microglia, with 268 genes identified as being angiogenic (GO:0001525), while 39 genes were involved in vascular constriction and 41 genes in vascular dilation (regulation of blood vessel diameter, GO:0097746). (H) Vessel diameter was quantified in rat retinal explants preincubated in Ames (black trace) or Ames + 230 nM candesartan (red trace) for 10 min, after which fractalkine (FKN, 200 ng/mL) was added (shaded area, representative data from 1 retina, n = 5 vessels). (I) When grouped data were analyzed 10 min after fractalkine addition, constriction was abolished when preincubated with candesartan (n = 7 fractalkine, n = 5 fractalkine + candesartan). Further supporting a role for the RAS, ex vivo incubation with fractalkine up-regulated microglial Agt expression, while this was not evident in microglia isolated from Cx3cr1^GFP/GFP retinae (Inset, n = 6). Data expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

Retinal blood flow is reduced and capillaries are constricted after 4 wk of diabetes. VFA was used to quantify retinal blood flow in control and STZ-treated animals. (A–C) En face heat-maps depicting fill time for arterioles, capillaries, and venules, with Insets showing representative average normalized fluorescence intensity traces for control (black line) and STZ-treated (red line) animals. (Scale bar, 500 μm in A.) (D and E) The times taken to reach half-maximum intensity (D, fill time) and half of final value from maximum (E, drain time) were quantified, showing fill and drain times were significantly increased in all vessel types in STZ-treated animals (unfilled bars, control n = 23; filled bars STZ, n = 21). (F) Sodium fluorescein fundus images were quantified for large vessel tortuosity (n = 13) and arteriovenous ratio (Inset, n = 13), with no difference observed between STZ-treated (filled bars) and control (unfilled bars) animals. (G) Immunohistochemistry was used to quantify vascular density in control and STZ-treated (unfilled and filled bars) eyes, with no difference observed between the two groups (n = 11). The rendered image shows the segmented vessel types (Inset, capillaries in yellow, arterioles in blue, and venules in cyan). (Scale bars, 1 mm.) (H) OCTA was performed in vivo to measure capillary diameter in control and STZ-treated (Inset) animals, with the vessels measured shown in green. (Scale bar, 50 μm.) (I) Decreased capillary diameter was observed in STZ-treated animals (n = 12) compared to control (n = 10) within the superior vascular plexus. No alteration was observed in the intermediate/deep vascular plexi. Group data expressed as mean ± SEM, *P < 0.05.

Fig. 4.

Microglia increase their contact with retinal capillaries after 4 wk of STZ-induced diabetes. (A) Whole-mounted retina from control (Inset) and STZ-treated animals were labeled for Iba-1 (microglia, green) and IB4-FITC (blood vessels, red) and the extent of microglial and vessel contact quantified for each vessel type. While no difference in large vessel contacts occurred, microglia–capillary contact increased in the central retina of the STZ-treated animals (filled bars, n = 11). (Scale bar, 50 μm.) (B) Control (Inset) and STZ-treated animals were labeled for Iba-1 (microglia, green), NG2 (pericytes, light blue), and IB4-FITC (blood vessels, red) and the extent of microglia–pericyte contact quantified for each vessel type. Microglial–pericyte association increased within the central retina of STZ-treated animals (filled bars, n = 11). (Scale bar, 50 μm.) (C) Using similar immunolabeling as in B, microglial association with pericyte somata, processes, and capillary areas lacking pericyte contact was quantified. The image analysis render (Inset) highlights pericyte somata (red), pericyte processes (green), and pericyte-free vessels (blue), while microglia touching each of these regions were skeletonized and color-coded for quantification. While there were no preferential association, all contacts were increased in STZ-treated (filled bars, n = 5) compared to control (unfilled bars, n = 5) retinae (STZ treatment P = 0.0015). (Scale bar, 50 μm.) (D and E) Macroglial change was assessed in control (unfilled bars, n = 11) and STZ-treated (filled bars, n = 11) retinae, with no alteration in astrocyte coverage (D), nor Müller cell gliosis (E) observed (n = 6). (F) VFA was used to quantify fluorescein offset as a measure of BRB integrity. While arterioles and venules showed no change, capillary offset was increased in STZ-treated animals (unfilled bars, control n = 23; filled bars STZ, n = 21). (G and H) The inflammatory status of microglia was assessed morphologically and no difference was found in the number of monocytes/microglia in central and peripheral retina (G, n = 11), cell soma size, mean process length, or process branching points (H, n = 5 control, n = 8 STZ). (I) RNA-seq data from retinal microglia taken from control and STZ-treated rats were screened for genes involved in the positive (GO:0050729) and negative (GO:0050728) regulation of inflammation. While some inflammatory genes were altered, key inflammatory genes were unchanged after 4 wk of diabetes. Data represented as mean ± SEM, *P < 0.05.

Fig. 5.

Vasoactive gene expression from retinal microglia and fractalkine-induced vasoconstriction are altered after 4 wk of STZ-induced diabetes. (A) The responsiveness of retinal vessels to hyperoxic challenge was explored in vivo using OCTA (Insets show OCTA images from baseline and after exposure to O₂). (Scale bar, 200 μm.) While hyperoxic challenge (filled bars) lead to constriction in the control group (n = 10 normoxia, n = 6 100% O₂), no constriction was observed in the STZ cohort (n = 12 normoxia, n = 7 100% O₂). (B) Microglial vasoregulation was investigated during diabetes, with 4-wk STZ-treated and control retinae exposed to fractalkine ex vivo (representative control and STZ images in Inset) (Scale bar, 50 μm.) While vessels from control retinae showed fractalkine-induced vasoconstriction (filled bar), STZ retinae exhibited no change (n = 5 animals). (C) Differential microglial gene expression data from 4 wk control and STZ-treated animals were compared to vasomodulatory gene lists (vasoconstriction, GO:0097746; angiogenesis, GO:0001525; vasodilation, GO:0097746), with the RAS positive regulator angiotensinogen (Agt), and negative regulator (Ahr) significantly altered (FDR-adjusted, citrate control n = 5, STZ n = 4). (D) OCTA was used to quantify retinal superficial capillary diameter in 4-wk control and STZ-treated animals (unfilled and filled bars, respectively) exposed to candesartan or vehicle. In STZ-treated animals, capillary diameter returned to baseline in the candesartan-treated group (n = 7 control, n = 8, 5 STZ vehicle and candesartan, respectively). (E) Retinal blood flow was quantified using arterio-venous transit time and showed increased transit time (slower blood flow) in STZ-treated animals independent of candesartan treatment (n = 8 control, n = 11 and 8 STZ vehicle and candesartan, respectively). (F) Quantification of the arteriovenous ratio showed candesartan treatment increased the diameter of larger vessels in STZ-treated retinae relative to control and vehicle-treated tissues (n = 8 control, n = 11 and 8 STZ vehicle and candesartan, respectively). Data expressed as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6.

Schematic representation of microglial regulation of retinal capillary constriction. Data from this study show microglia are structurally and functionally capable of involvement in the neurovascular unit. Microglia contact neuronal synapses and retinal capillaries (including pericytes) and activation of fractalkine-Cx3cr1 signaling results in capillary constriction, which is via an AT1R-dependent mechanism. Ultimately, capillary regulation may occur via direct microglial mechanism or may involve contributions from pericytes and Müller cells. EC, endothelial cell; PC, pericyte.