Table 1 Impact acceleration condition and survival time for each experimental group
Experiments/groups | Experimental history, survival times post-IA | Procedures |
|---|---|---|
1.Characterization of initial injury in the optic nerve - general | Sham (n = 3), 4 h post-IA (n = 3), 24 h post-IA (n = 3) IA:40 g × 1 m or 60 g × 0.85 m | IHC for APP (TAI) and IgG (BBB disruption) |
2. Characterization of initial injury in the optic nerve - CLARITY | CTB injection immediately post-IA, survival 2 d post-IA (n = 3) IA: 40 g × 1 m -– 60 g × 0.85 m | CLARITY |
3. Characterization of traumatic axonopathy - general neuropathology in the optic nerve and tract | 7 d post-IA (n = 3) IA:40 g × 1 m or 60 g × 0.85 m | Gallyas silver IHC for neuroinflammation (IBA1) |
4. Characterization of traumatic axonopathy - death of RGCs in retina | Sham (n = 6), 2 wk. post-IA (n = 7), 4 wk. post-IA (n = 5) IA:40 g × 1 m | IHC for γ Synuclein Cell counts on retinal wholemounts |
5. Characterization of traumatic axonopathy - axonal degeneration in optic nerve | Sham (n = 6), 2 wk. post-IA (n = 7), 4 wk. post-IA (n = 5) IA:40 g × 1 m | Embedding of optic nerve tissues in epoxy resin, semithin sectioning, toluidine blue staining |
6. Induction of DLK-JNK pathway in RGCs after injury | 24 h (n = 3), 3 d post-IA (n = 3) IA:40 g × 1 m | IHC for DLK, p-JUN in retinal sections |
7. Time course of DLK-JNK induction in RGCs after injury | Sham (n = 4), 24 h post-IA (n = 10), 3 d post-IA (n = 5), 7 d post-IA (n = 4), 14 d post-IA (n = 3) IA:40 g × 1 m | IHC for γ Synuclein and p-JUN Cell counts on retinal wholemounts |
8. Time course of activation of distinct member of DLK-JNK pathway in RGCs after injury | Sham (n = 3), 24 h post-IA (n = 3), 3 d post-IA (n = 3), 7 d post-IA (n = 3) IA:40 g × 1 m | Standard Western blotting |
9. Interventional studies - genetic deletion of Dlk on Dlkfl/fl mice and effects on DLK-JNK pathway activation and RGC survival | AAV2-Cre-GFP into one eye AAV2-GFP into fellow eye (n = 4 each, injections 2 wk. pre-IA, euthanasia 3 days post-IA) IA:60 g × 0.85 m | IHC for p-JUN Cell counts on retinal wholemounts |
AAV2-Cre-GFP into one eye AAV2-GFP into fellow eye (n = 7 each, injections 2 wk. pre-IA, euthanasia 4 wk. post-IA) IA:60 g × 0.85 m | IHC for RBPMS Cell counts on retinal wholemounts | |
10. Interventional studies - genetic deletion of Dlk and Lzk on Dlkfl/flLzkfl/fl and effects on DLK-JNK pathway activation and RGC survival | AAV2-Cre-GFP into one eye AAV2-GFP into fellow eye (n = 4 each, injections 2 wk. pre-IA, euthanasia 3 day post-IA) IA:60 g × 0.85 m | IHC for p-JUN Cell counts on retinal wholemounts |
AAV2-Cre-GFP into one eye AAV2-GFP into fellow eye (n = 7 each, injections 2 wk. pre-IA, euthanasia 4 wk. post-IA) IA:60 g × 0.85 m | IHC for RBPMS Cell counts on retinal wholemounts | |
11. Interventional studies - pharmacologic kinase inhibition with sunitinib and effects on DLK-JNK pathway activation and RGC survival | Drug (n = 12) Vehicle (n = 14), both groups 3 wk. post-IA IA:40 g × 1 m | IHC for γ Synuclein Cell counts on retinal wholemounts |