Journal: Proceedings of the National Academy of Sciences of the United States of America

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

doi: 10.1073/pnas.2112561118

Figure Lengend Snippet: 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 expo- sure 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 con- trol 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.

Article Snippet: Recombinant rat fractalkine (200 ng/mL; R&D Systems, #537-FT-025/CF) or vehicle (PBS) was introduced after 10 min of baseline recording and imaged for an additional 10 min. At the end of this incubation, vessel diameter was measured at sites with and without microglial contact (2 to 4 individual capillary sites per retina, n = 4 to 6 retinae) and measurements expressed as a percentage of baseline diameter of the same vessel region (taken as the average vessel diameter over the initial 10-min baseline).

Techniques: Gene Expression, In Vivo, Control, Ex Vivo

Journal: Proceedings of the National Academy of Sciences of the United States of America

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

doi: 10.1073/pnas.2112561118

Figure Lengend Snippet: Fig. 6. Schematic representation of microglial regulation of ret- inal capillary constriction. Data from this study show microglia are structurally and functionally capable of involvement in the neurovascular unit. Microglia contact neuronal synapses and reti- nal 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 contri- butions from pericytes and M€uller cells. EC, endothelial cell; PC, pericyte.

Article Snippet: Recombinant rat fractalkine (200 ng/mL; R&D Systems, #537-FT-025/CF) or vehicle (PBS) was introduced after 10 min of baseline recording and imaged for an additional 10 min. At the end of this incubation, vessel diameter was measured at sites with and without microglial contact (2 to 4 individual capillary sites per retina, n = 4 to 6 retinae) and measurements expressed as a percentage of baseline diameter of the same vessel region (taken as the average vessel diameter over the initial 10-min baseline).

Techniques: Activation Assay

Journal:

Article Title: Expression of fractalkine and fractalkine receptor in urinary bladder after cyclophosphamide (CYP)-induced cystitis

doi: 10.1016/j.autneu.2006.02.030

Figure Lengend Snippet: A. Representative example of a western blot of whole urinary bladder (20 μg) for fractalkine expression in control rats and those treated with cyclophosphamide (CYP) for varying duration. Actin staining was used as a loading control and recombinant rat fractalkine/CX3CL1 (2.5 ng) was used as a positive control. B. Histogram of relative fractalkine band density in all groups examined (n = 8 for each) normalized to actin staining presented as a percentage of control fractalkine expression. Fractalkine receptor expression in urinary bladder is significantly increased at all time points of CYP-induced cystitis examined. *, p ≤ 0.01.

Article Snippet: Recombinant rat fractalkine/ CX3CL1 (2.5 ng; R&D Systems, Minneapolis, MN) was used as a positive control and western blotting of actin (1:20,000; Santa Cruz, Santa Cruz, CA) was used as a loading control.

Techniques: Western Blot, Expressing, Control, Staining, Recombinant, Positive Control