Post-injury born oligodendrocytes incorporate into the glial scar and contribute to the inhibition of axon regeneration

**Background:** Failure of CNS projection neurons to regenerate long-distance axons after white matter injury leads to permanent disability. Even when intrinsic regenerative mechanisms are stimulated (e.g., Pten knockdown), regenerating axons stall before reaching postsynaptic targets. The authors hypothesized that live oligodendrocytes—which are absent during developmental axon growth but present after injury—contribute to this stalling, beyond the known inhibitory effects of myelin debris.

**Methods:** Optic nerve crush (ONC) was performed in adult male 129S1/SvlmJ mice. To label newly born cells, EdU was injected daily from day 3 to day 10 post-ONC. Single-cell RNA-seq (scRNA-seq) was performed on cells from uninjured and injured (2 weeks post-ONC) optic nerves. For demyelination, mice received a cuprizone diet (0.3% decreasing to 0.1%) plus intraperitoneal rapamycin (10 mg/kg, 5 days/week) for 6 weeks before ONC, with rapamycin stopped 2 weeks before ONC. AAV2 expressing anti-Pten shRNA or scrambled control was injected intravitreally 2 weeks before ONC. Axon regeneration was assessed 2 weeks post-ONC by CTB tracing. In separate experiments, a single intravitreal injection of cuprizone (100 µM, 1 mM, or 10 mM) was given immediately after ONC. RGC survival was quantified by RBPMS immunostaining. Myelin and oligodendrocyte markers (MBP, CC1) were measured by immunofluorescence. Microglia/macrophage activation was assessed by Iba1 staining and fibrotic scarring.

**Key Results:** - One day after ONC, TUNEL+ cells and Olig2+ debris were abundant in the injury site; by 2 weeks, the injury site was repopulated with Olig2+/CC1+ oligodendrocyte lineage cells to pre-injury density. EdU labeling showed a nearly fivefold increase in newborn EdU+/Olig2+ cells in the injury site compared with uninjured controls (P<0.001). - scRNA-seq revealed a 14-fold increase in immune cells (Iba1+/CD45+/C1qc+) at the injury site 2 weeks post-ONC. Oligodendrocyte lineage analysis showed a decrease in mature oligodendrocytes (MOs) and an increase in newly formed oligodendrocytes (NFOs) and a novel population of MBP mRNA+/S100β+/MAG− small spidery oligodendrocytes (sSOs) that were nearly absent in uninjured nerves. - The demyelination regimen reduced CC1+ oligodendrocytes in the injury site (P<0.001) but did not affect Pdgfra+ OPCs or CSPG labeling. MBP signal decreased by 10% (P<0.01) in the uninjured optic nerve and was nearly absent in the injury site even without cuprizone. - Pten KD alone stimulated axon regeneration; combining Pten KD with the demyelination regimen significantly increased the number of regenerating axons at distances up to 3 mm from the injury site (overall F=8.0, P<0.001; pairwise comparisons in Table S2A). RGC survival was modestly improved in the combination group (P<0.02). - Localized intravitreal cuprizone (1 mM) also promoted axon regeneration (overall F=2.9, P<0.03; Table S2B), without activating microglia/macrophage or causing fibrotic scarring, unlike zymosan (P<0.01 for Iba1 number and size). - In vitro, cuprizone did not affect RGC survival or axon growth at concentrations up to 10 mM. - scRNA-seq showed that post-injury born NFOs and sSOs express axon growth-inhibitory membrane proteins: NogoA (Rtn4), Omgp (Omg), Mag, BAI1 (Adgrb1), BAI2 (Adgrb2), and Sema5A. Expression levels varied by cell type and injury condition (e.g., Rtn4 was significantly higher in NFOs and MOs after injury, P<0.01).

**Clinical Implications:** This study identifies live, post-injury born oligodendrocytes within the glial scar as a novel cellular barrier to CNS axon regeneration. The finding that cuprizone-induced reduction of these cells enhances regeneration suggests that therapeutic strategies temporarily suspending oligodendrocyte–axon interactions could allow regenerating axons to reach their targets. The scRNA-seq resource (Subtype Gene Browser) provides a platform for further investigation of oligodendrocyte subtypes in injury and repair. Future work should explore localized, non-toxic methods to transiently inhibit oligodendrocyte-mediated inhibition without causing demyelination.