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: Frontiers in Cellular Neuroscience

Article Title: CX 3 CL1 is up-regulated in the rat hippocampus during memory-associated synaptic plasticity

doi: 10.3389/fncel.2014.00233

Figure Lengend Snippet: Expression of CX 3 CL1 in the CA1, CA3 and dentate gyrus (DG) of the rat hippocampus. (A) Basal levels of CX 3 CL1 expression in the rat dorsal hippocampus. Green: CX 3 CL1; Red: propidium iodide-stained nuclei. Scale bar = 200 μm. (B–D) CA1, CA3 and DG regions of the hippocampus, respectively, showing CX 3 CL1 (green), NeuN (red) and Hoechst (blue) expression. White arrowheads: interneurons; yellow arrowheads: CA pyramidal neurons. (E) Relationship between CX 3 CL1 and NeuN expression. Each dot represents a single cell. Linear regression analyses (red lines) appear on each plot with corresponding r 2 values indicated. Linear regression p < 0.05 for CA1, CA3 and DG regions of the hippocampus.

Article Snippet: The effect of 500 pM rCX 3 CL1 (recombinant rat CX 3 CL1, Peprotech EC) on LTP in the DG was investigated both in the presence and absence of 100 μM of the GABA A receptor/chloride channel inhibitor, picrotoxin (Sigma–Aldrich).

Techniques: Expressing, Staining

Journal: Frontiers in Cellular Neuroscience

Article Title: CX 3 CL1 is up-regulated in the rat hippocampus during memory-associated synaptic plasticity

doi: 10.3389/fncel.2014.00233

Figure Lengend Snippet: Temporal changes in CX 3 CL1 expression in the rat hippocampus post-spatial learning measured by immunofluorescence. (A) Graph shows the average latency-to-platform times for rats trained in a single session (five trials) of the water maze task ( n = 12). (B–D) Box plots represent the distributions of fluorescence intensity values for CX 3 CL1 protein expression in CA1, CA3, and DG cells at 1, 2 and 3 h post-water maze training, compared to passive control animals. Changes in CX 3 CL1 expression were analyzed using Kruskal–Wallis ANOVA and Dunn’s multiple comparisons post hoc tests; ∗ p < 0.01. There were four rats per time-point and three hippocampal sections analyzed per animal. The numbers of cells analyzed per hippocampal region were as follows: CA1: 1116–1201 cells; CA3: 1090–1247 cells; DG: 2888–3455 cells. The numbers of cells analyzed per time-point were: 1 h: 11,526 cells; 2 h: 10,355 cells; 3 h: 10,867 cells.

Article Snippet: The effect of 500 pM rCX 3 CL1 (recombinant rat CX 3 CL1, Peprotech EC) on LTP in the DG was investigated both in the presence and absence of 100 μM of the GABA A receptor/chloride channel inhibitor, picrotoxin (Sigma–Aldrich).

Techniques: Expressing, Immunofluorescence, Fluorescence, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: CX 3 CL1 is up-regulated in the rat hippocampus during memory-associated synaptic plasticity

doi: 10.3389/fncel.2014.00233

Figure Lengend Snippet: CX 3 CL1 expression increases 2 h following theta-burst stimulation-induced LTP in the DG of acute hippocampal slices. (A) Schematic diagram illustrating the position of stimulating and recording electrodes in hippocampal LTP experiments. Electrodes were always placed in the medial molecular layer of the lower blade of the DG approximately 100–200 μm apart. US, unstimulated DG blade; TBS, theta-burst stimulated blade. (B) Box plots showing the distribution of CX 3 CL1 fluorescence intensities in the upper and lower blades of the DG in stimulated slices and time-matched control slices. Asterisk indicates significant difference from the US blade (Kruskal–Wallis ANOVA and Dunn’s multiple comparisons post hoc tests; p < 0.001).

Article Snippet: The effect of 500 pM rCX 3 CL1 (recombinant rat CX 3 CL1, Peprotech EC) on LTP in the DG was investigated both in the presence and absence of 100 μM of the GABA A receptor/chloride channel inhibitor, picrotoxin (Sigma–Aldrich).

Techniques: Expressing, Fluorescence, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: CX 3 CL1 is up-regulated in the rat hippocampus during memory-associated synaptic plasticity

doi: 10.3389/fncel.2014.00233

Figure Lengend Snippet: Differential effects of CX 3 CL1 on long-term potentiation (LTP) in the DG and glutamate-induced intracellular calcium rise in the CA1, in the presence and absence of GABA A /chloride receptor blockade. (A) The effect of the chemokine domain of CX 3 CL1 on LTP in acute hippocampal slices as measured by augmented field EPSP magnitude in the DG following theta-burst stimulation (TBS: 8 × 8 × 200 Hz). CX 3 CL1 inhibited dentate LTP in both the early and late phases post-TBS (i.e., 20–40 min and 40–80 min, respectively; one-way ANOVA; p < 0.05, indicated by an asterisk; n = 8 slices per group). CX 3 CL1 (500 pM) was present for the duration of the time period shown. (B) Shows the paired-pulse ratio between the first and second stimulations (50 ms interval) in the LTP experiment in A . (C) Shows the effect of CX 3 CL1 on dentate LTP in acute hippocampal slices as measured by augmented field EPSP magnitude following TBS in the presence of picrotoxin (100 μM). CX 3 CL1 (500 pM) and picrotoxin were present for the duration of the time period shown. CX 3 CL1 enhanced early LTP (one-way ANOVA; p < 0.05, indicated by an asterisk; n = 8 slices per group) while having no effect on late LTP. (D) Shows the paired-pulse ratio between the first and second stimulations (50 ms interval) in the LTP experiment in C . (E) Shows the effect of CX 3 CL1 on glutamate-induced calcium influx in the CA1 region of organotypic hippocampal slices cultured for 21 DIV. Pre-treatment of slice cultures with CX 3 CL1 (500 pM) for 15 min prior to glutamate exposure reduced calcium influx in the CA1 region (Mann–Whitney U test; p < 0.001). Box plot inset shows the area under the curve (AUC) for the whole experimental time-course. CX 3 CL1 (500 pM) was present for the duration of the time period shown. (F) The effect of CX 3 CL1 on glutamate-induced calcium influx in the CA1 region of organotypic hippocampal slices in the presence of picrotoxin. Pre-treatment of slice cultures with CX 3 CL1 (500 pM) and picrotoxin (100 μM) versus picrotoxin alone (control) for 15 min prior to glutamate exposure enhanced calcium influx in the CA1 region (Mann–Whitney U test; p < 0.001). Box plot inset shows the area under the curve (AUC) for the whole experimental time-course. CX 3 CL1 (500 pM) and/or picrotoxin (100 μM) were present for the duration of the time period shown.

Article Snippet: The effect of 500 pM rCX 3 CL1 (recombinant rat CX 3 CL1, Peprotech EC) on LTP in the DG was investigated both in the presence and absence of 100 μM of the GABA A receptor/chloride channel inhibitor, picrotoxin (Sigma–Aldrich).

Techniques: Cell Culture, MANN-WHITNEY, Control

Journal: Frontiers in Cellular Neuroscience

Article Title: CX 3 CL1 is up-regulated in the rat hippocampus during memory-associated synaptic plasticity

doi: 10.3389/fncel.2014.00233

Figure Lengend Snippet: Effect of CX 3 CL1 on glutamate-induced calcium dynamics in neurons and glia. (A–C) Representative images of intracellular calcium [Ca 2+ ] i levels in mixed neuron-glial primary hippocampal cultures before and after 30 μM glutamate exposure. Control cells (A) were untreated prior to glutamate exposure. Treated cells were exposed to either (B) 500 pM or (C) 2 nM CX 3 CL1 for 15 min. Scale bar = 200 μm. (D,E) Shows the time-course of the [Ca 2+ ] i response to glutamate in neurons (D) and non-neuronal cells (E) . Cells were treated for 15 min with either 500 pM (blue circles) or 2 nM (green circles) CX 3 CL1 for 15 min prior to 30 μM glutamate exposure. Untreated control time-course is represented by black circles. Relative changes in [Ca 2+ ] i were calculated for each cell at each time point as f / f 0 , where f is the [Ca 2+ ] i fluorescence in each frame and f 0 is the average baseline fluorescence per cell, calculated 20 s prior to glutamate addition. Primary hippocampal cell cultures were divided into neurons (138, 171, and 101 cells for control, 500 pM and 2 nM CX 3 CL1-treated cells, respectively) and non-neuronal (432, 726, and 604 cells for control, 500 pM and 2 nM CX 3 CL1-treated cells, respectively) cell populations based on their [Ca 2+ ] i response to 30 μM glutamate . (F,G) Quantification of the calcium imaging time-courses in D and E . 500 pM CX 3 CL1 attenuates glutamate-induced [Ca 2+ ] i increases in non-neuronal cell types (Kruskal–Wallis ANOVA and Dunn’s multiple comparisons post hoc tests; ∗ p < 0.05), but has no significant effect on [Ca 2+ ] i response in neurons. Pre-treatment of hippocampal cell cultures for 15 min with 2 nM CX 3 CL1, however, significantly attenuates glutamate-induced [Ca 2+ ] i influx in both non-neuronal cells and in neurons (Kruskal–Wallis ANOVA and Dunn’s multiple comparisons post hoc tests; ∗ p < 0.001). The results represent combined data from five to six individual cover-slips per treatment group and from two separate cell culturing days.

Article Snippet: The effect of 500 pM rCX 3 CL1 (recombinant rat CX 3 CL1, Peprotech EC) on LTP in the DG was investigated both in the presence and absence of 100 μM of the GABA A receptor/chloride channel inhibitor, picrotoxin (Sigma–Aldrich).

Techniques: Control, Fluorescence, Imaging, Cell Culture

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