Visually induced changes in cytokine production in the chick choroid
- Department of Cell Biology, University of Oklahoma Health Sciences Center, United States
Figures
Figure 1 with 2 supplements
Immunohistochemical localization of IL-6 in chick choroids.
(A, B) Il-6 was localized in treated and contralateral control eyes after 24 hr of recovery from induced myopia (green labeling). (C) Preabsorption of anti-IL-6 with a tenfold molar excess of …
Figure 1—figure supplement 1
Immunolocalization of IL-6 in the chick choroid.
(A, B) Adjacent longitudinal sections (10 μm thick) of control chick choroids following standard H&E staining (A), and immunolabeling for IL-6 (B). (C, D) Adjacent oblique sections through chick …
Figure 1—figure supplement 2
Improved visualization of DAPI labeling in chick RPE and choroid.
DAPI-labeled nuclei in RPE and choroid from Figure 1 were visualized by increasing the DAPI channel (405 nm) intensity. RPE nuclear layer is located between dotted lines in (A–C). IL-6 …
Figure 2 with 1 supplement
Microarray identifies IL-6 as a gene highly overexpressed in early recovery.
A volcano plot of Affymetrix chicken microarray data indicated that 207 genes were found to be significantly differentially expressed by ≥2-fold in recovering choroids as compared with choroids from …
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Figure 2—source data 1
Microarray data.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Refractions of form-deprived (FD; right eyes) and untreated contralateral control eyes (left eyes) following 10 days of form deprivation.
Monocular form-deprivation of chicks in this study resulted in a large negative shift in refractive error. Data are represented as mean ± standard errors. *p=0.0313 Wilcoxon matched-pairs …
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Figure 2—figure supplement 1—source data 1
Refractions of form-deprived and untreated contralateral control eyes.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig2-figsupp1-data1-v2.xlsx
Figure 3 with 1 supplement
Cytokine gene and protein expression in chick choroids.
(A) IL-6 mRNA expression in choroids from control and treated eyes, following 10 days of form deprivation (0 hr/10 days FD), 0.75 hr to 8 days of recovery from form deprivation, normal, untreated …
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Figure 3—source data 1
Cytokine gene and protein expression in chick choroids.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
IL-6 has no direct effect on scleral proteoglycan synthesis.
Incubation of chicken sclera in organ culture with recombinant chicken IL-6 (10 ng/ml) for 24 hr had no significant effect on scleral proteoglycan synthesis (p=0.1439, Student’s t-test, n=11 sclera …
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Figure 3—figure supplement 1—source data 1
IL-6 on scleral proteoglycan synthesis.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig3-figsupp1-data1-v2.xlsx
Figure 4 with 1 supplement
Effect of light intensity on IL-6 mRNA expression.
Normal chicks were housed in complete darkness (dark), white LED light of varying intensities (‘low,’ 5 lux; ‘medium,’ 700 lux; ‘high,’ 3150 lux), red LED light (‘red,’ 58 lux), or blue LED light …
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Figure 4—source data 1
Results of Taqman (IL-6/GAPDH ∆∆C(t)) Light Intensity Experiment.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Spectra of LED light sources as a function of wavelength.
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Figure 4—figure supplement 1—source data 1
Spectra of LED light sources as a function of wavelength.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig4-figsupp1-data1-v2.xlsx
Figure 5
Effect of imposed defocus on choroidal IL-6 gene expression.
(A). Spectacle lenses [minus 15 D (–15) or plus 15 D (+15)] were applied to the right eyes of chicks for 6–24 hr. (B). Schematic diagram illustrating the effects of imposed optical defocus on the …
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Figure 5—source data 1
Effect of imposed defocus on choroidal IL-6 gene expression.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig5-data1-v2.xlsx
Figure 6
L-NAME inhibits choroidal IL-6 transcription and recovery.
(A) Intravitreal injection of L-NAME (16.2 μmol/eye) immediately prior to recovery significantly reduced IL-6 mRNA levels compared to recovering eyes receiving vehicle only (0.9% NaCl) (**p=0.007, …
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Figure 6—source data 1
The effect of L-NAME on choroidal IL-6 transcription and recovery.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig6-data1-v2.xlsx
Figure 7
The NO donor, PAPA-NONOate, stimulates choroidal IL-6 production.
Choroids were isolated from normal chicken eyes and were incubated with the indicated concentrations of PAPA-NONOate for 24 hr. (A) IL-6 gene expression was significantly increased in choroids …
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Figure 7—source data 1
The effect of the NO donor, PAPA-NONOate on choroidal IL-6 production.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig7-data1-v2.xlsx
Figure 8
L-arginine (L-arg), the NOS substrate, stimulates choroidal IL-6 trasncription.
Choroids were isolated from normal chicken eyes and were incubated with the indicated concentrations of L-arg for 24 hr. KCl (50 mM) was added to some cultures to depolarize cell membranes. (A) IL-6 …
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Figure 8—source data 1
The effect of L-arginine on choroidal IL-6 transcription.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig8-data1-v2.xlsx
Figure 9
Atropine stimulates choroidal IL-6 gene expression.
(A) Intravitreal injection of atropine (240 nmol/eye) into chick eyes following 14 days of form deprivation (myopic eyes) increased IL-6 mRNA levels compared to myopic eyes receiving vehicle only …
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Figure 9—source data 1
The effect of atropine on choroidal IL-6 gene expression.
- https://cdn.elifesciences.org/articles/70608/elife-70608-fig9-data1-v2.xlsx
Figure 10
Proposed role of IL-6 in the retina-to-sclera signaling cascade.
Myopic defocus initiates a series of signaling events in the retina potentially involving dopamine, nitric oxide synthase (NOS), and nitric oxide (NO) (as well as other mediators not shown). Nitric …
Tables
Key resources table
| Reagent type(species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-chick IL-6(Rabbit polyclonal) | Bio-RadLaboratories | Cat#: AHP942ZRRID: AB_2127753 | IF (1:20) |
| Sequence-based reagent | Chicken IL-6Taqman GeneExpression Assay | Thermo FisherScientific | Gg03337980_m1unlabeled PCR primers andFAM-labeled TaqMan probe | |
| Sequence-based reagent | Chicken interferon γTaqMan GeneExpression Assay | ThermoFisherScientific | Gg03348618_m1unlabeled PCR primers andFAM-labeled TaqMan probe | |
| Sequence-based reagent | Chicken IL-1βTaqMan GeneExpression Assay | Thermo FisherScientific | Gg03347154_g1unlabeled PCR primers andFAM-labeled TaqMan probe | |
| Sequence-based reagent | Chicken TNF-α(LITAF) TaqMan GeneExpression Assay | Thermo FisherScientific | Gg03364359_m1 unlabeled PCR primers andFAM-labeled TaqMan probe | |
| Sequence-based reagent | Chicken GAPDHTaqMan GeneExpression Assay | Thermo FisherScientific | Gg03346982_m1unlabeled PCR primers andFAM-labeled TaqMan probe | |
| Peptide, recombinant protein | Chicken IL-6 | Bio-RadLaboratories | Cat#: PAP003 | |
| Commercial assay or kit | High capacityRNA-to-cDNA kit | AppliedBiosystems | Cat#: 4388950 | |
| Commercial assay or kit | DNase treatment& removal kit | Invitrogen | Cat#: AM1906 | |
| Chemical compound, drug | SB 203580 | Sigma-Aldrich | Cat#: S8307 | |
| Chemical compound, drug | Atropine(Sulfate Salt) | Sigma-Aldrich | Cat#: A-0257 | |
| ChemicalCompound, drug | L-NAME | Sigma-Aldrich | Cat#: N5751 | |
| Chemical compound, drug | PAPA-NONOate | CaymanChemical | Cat#: 82,140 | |
| Other | DAPI stain | Invitrogen | Cat#: D3571 | (5 µg/ml) |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70608/elife-70608-transrepform1-v2.docx
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Supplementary file 1
Affymetrix microarray analysis of gene expression in control and recovering chick choroids.
- https://cdn.elifesciences.org/articles/70608/elife-70608-supp1-v2.xlsx
