Additional file 2 of AMPK hyperactivation promotes dendrite retraction, synaptic loss, and neuronal dysfunction in glaucoma
Bibliographic record
Abstract
Additional file 2: Supplementary Figure 1. (A) The number of YFP-positive RGC does not change at 2 weeks of ocular hypertension (OHT) relative to non-injured controls (Student’s t-test, n.s.: not significant, N = 5 mice/group). (B) Real-time qPCR analysis confirms that retinal YFP gene expression does not change within 2 weeks of OHT damage (Student’s t-test, n.s.: not significant, N = 5 mice/group). (C-E) RGC co-expressing YFP and SMI-32 are selected for dendritic arbor imaging and 3D reconstruction. Supplementary Figure 2. Analysis of whole-mounted retinas immunolabeled with an antibody recognizing pAMPKThr172 revealed a substantial increase of AMPK activity in RGC axons, visualized with SMI-32, at two weeks after glaucoma induction. Scale bars = 25 μm. Supplementary Figure 3. (A, B) Retinal imaging showed no fluorescein extravasation in eyes injected with compound C or vehicle. (C) In contrast, marked fluorescein extravasation was found in ischemic retinas subjected to central retinal artery ligature. (D), a procedure known to compromise the BRB [31] and used as positive control (Fig. S3C). (D, E) T cell infiltration, visualized with markers the MHCII, MPO and CD45, was not detected in retinas treated with compound C or vehicle. (F) Ischemic retinas, used as positive controls, display cells positive for MHCII, MPO and CD45. (Q) Quantification of cells expressing MHCII, MPO and CD45 confirm lack of cellular infiltrates in compound c-treated retinas. (ANOVA with Tukey’s multiple comparison post-hoc test, * = p<0.05, N = 3 mice/group). Scale bars = 10 μm. Values are expressed as the mean ± S.E.M. Supplementary Figure 4. (A) Intravitreal delivery of non-targeting (scrambled) Cy3-tagged control siRNA (siCTL-Cy3) results in rapid uptake by neurons in the ganglion cell layer (GCL), (B-D) identified as RBPMS-positive RGC (B, inset in A) as early as 3 h after injection. Scale bars (A) = 20 μm, (B-D) = 20 μm. OPL: Outer Plexiform Layer, INL: Inner Nuclear Layer, IPL: Inner Plexiform Layer. Supplementary Figure 5. (A) In the inner nuclear layer (INL), pS6Ser240/244 co-localizes with the calcium-binding protein calbindin, a marker of horizontal cells. (B) pS6Ser240/244 expression in horizontal cells remains unchanged after OHT induction (Student’s t-test, n.s.: not significant, N = 5 mice/group). (C, D) Immunohistochemical analysis of PSD95 and VGLUT1 shows no appreciable change in excitatory synapses in sham retinas treated with siAMPK and vehicle or rapamycin (ANOVA with Tukey’s multiple comparison post-hoc test, n.s. = not significant, N = 3 mice/group). (E) The total area of sham-operated versus glaucomatous retinas is similar, ruling out changes caused by ocular hypertension (Student’s t-test, n.s = not significant, N = 5–6 mice/group). Scale bars (A) = 20 μm, (C) = 10 μm. Values are expressed as the mean ± S.E.M. IPL: Inner Plexiform Layer, GCL: Ganglion Cell Layer. Supplementary Figure 6. (A) Immunohistochemistry of optic nerve head (ONH) cross-sections shows that although Iba1-positive cells and GFAP increase with high intraocular pressure, siAMPK treatment did not alter this response relative to control siRNA (siCTL). (B) Quantification of Iba1-positive cells and GFAP fluorescence confirmed that siAMPK does not have a significant effect on the number of Iba1-positive cells or GFAP reactivity (ANOVA with Tukey’s multiple comparison post-hoc test, * = p<0.05, ** = p<0.01, N = 3 mice/group). Scale bars = 50 μm. Values are expressed as the mean ± S.E.M.
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How this classification was reachedexpand
Direct model labels (unvalidated)
Per-model category and study-design labels from the labeling rounds. They are machine output, unvalidated, and the disagreement between models ships as data. No study design here is MEDLINE-validated yet.
| Model arm | Categories | Study design | Confidence |
|---|---|---|---|
| gemma | no category Domain: not available · Genre: Dataset About the Canadian research system: no · About a Canadian topic: no | Not applicable | high |
| gpt | Insufficient payload (model declined to judge) Domain: not available · Genre: Dataset About the Canadian research system: no · About a Canadian topic: no | Not applicable | high |
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.024 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.002 | 0.003 |
| Science and technology studies | 0.001 | 0.000 |
| Scholarly communication | 0.003 | 0.002 |
| Open science | 0.002 | 0.001 |
| Research integrity | 0.002 | 0.001 |
| Insufficient payload (model declined to judge) | 0.912 | 0.194 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedLabeled directly by 2 models reading the full record.
The models disagree on parts of this classification; every voice is preserved in the section at the end of the page.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".