Re-establishing the regenerative potential of central nervous system axons in postnatal mice

Abstract

At a certain point in development, axons in the mammalian central nervous system lose their ability to regenerate after injury. Using the optic nerve model, we show that this growth failure coincides with two developmental events: the loss of Bcl-2 expression by neurons and the maturation of astrocytes. Before postnatal day 4, when astrocytes are immature, overexpression of Bcl-2 alone supported robust and rapid optic nerve regeneration over long distances, leading to innervation of brain targets by day 4 in mice. As astrocytes matured after postnatal day 4, axonal regeneration was inhibited in mice overexpressing Bcl-2. Concurrent induction of Bcl-2 and attenuation of reactive gliosis reversed the failure of CNS axonal re-elongation in postnatal mice and led to rapid axonal regeneration over long distances and reinnervation of the brain targets by a majority of severed optic nerve fibers up to 2 weeks of age. These results suggest that an early postnatal downregulation of Bcl-2 and post-traumatic reactive gliosis are two important elements of axon regenerative failure in the CNS.

Fig. 1

Robust and rapid optic nerve regeneration in P3 Bcl-2tg mice. Photomicrograph montages of adjacent longitudinal optic nerve sections and confocal epifluorescence photomicrographs show axonal morphology of wt (A,B) and Bcl-2tg (C-G) mice at 24 (A-D) and 48 hours (E-G) after optic nerve crush. The sections revealed CTB-R labeling (A,C) or were stained with antibodies against NF-M (B,D-G). F and G show highermagnification views of axon and growth cone morphologies (100×). Asterisks indicates the crush site; arrowheads indicate growth-cone-like structures. Bar, 100 μm (A-C); 40 μm (D,E); 10 μm (F,G).

Fig. 2

The majority of RGC axons in Bcl-2tg mice regenerate and reach the ipsilateral brain targets within 4 days. (A-F) Epifluorescence photomicrographs of coronal brain sections from a Bcl-2tg mouse on day 4. Note green fluorescence (CTB) in the ipsilateral SC, pretectal nuclei (PT) (A), dorsal (dLGN) and ventral LGN (vLGN) (B), and the optic tract (C). Weak fluorescence is present in the corresponding contralateral targets (D-F). Dotted lines outline the SC and dLGN. Arrows indicate regenerating axons showing positive fluorescence in the optic tract. Bar, 200 μm. (G,H) Schematic drawings of CTB-labeled retinofugal projections in coronal sections of mouse brain. (G) Retinofugal projection formed by regenerating axons in Bcl-2tg mice. (H) Retinofugal projections in normal wt and Bcl-2tg mice. No apparent abnormality of retinal axon projection is noted in uninjured Bcl-2tg mice compared with wt controls.

Fig. 3

Quantitative assessment of axonal regeneration. (A-D) Photomicrographs of FluoroGold-labeled RGCs in wholemount retinas from injured and uninjured wt and Bcl-2tg mice on day 11 after optic nerve crush. Bar, 50 μm. (E) Number of retrogradely labeled RGCs in the retinal whole-mounts of wt and Bcl-2tg mice that underwent optic nerve crush (injured) or a sham procedure. (F) Distance of axonal regeneration on days 1–4. Values are mean±s.e.m.

Fig. 4

Onset of optic nerve regenerative failure in P5 Bcl-2tg mice coincides with astrocyte maturation. (A,B) Quantification of retinal axon regrowth in retina-midbrain slice co-cultures. Values are mean±s.e.m. (**P<0.01; ***P<0.001). (C) Photomicrograph montages of adjacent longitudinal optic nerve sections at day 4 after optic nerve crush in a P5 wt mouse and a Bcl-2tg mouse. The asterisk indicates the crush site. Bar, 250 μm. (D,E) Western blot analysis (D) and RT-PCR (E) reveal developmental expression patterns of myelin/oligodendrocyte-associated proteins and astrocyte markers in E14-P14 mouse midbrains. (F) Western blot analysis of GFAP expression in P0 and P5 normal (−) and injured (+) midbrain tissues. (G) Western blot analysis confirms the absence of MBP and MAG in the midbrains of jimpy mice. (H) Quantification of retinal axon regrowth into midbrain slices of P14 wt and jimpy mice in retina-midbrain slice co-cultures. Values are mean±s.e.m. (P=0.6).

Fig. 5

Robust optic nerve regeneration in adult Bcl-2tg mice after treatment with astrotoxin. (A-H) Photomicrograph montages of adjacent longitudinal optic nerve sections on day 8 after optic nerve crush in adult wt and Bcl-2tg mice. The asterisk indicates the crush site. Bar, 250 μm. (I,J) Electron micrographs (EM) of optic nerve sections collected at 0.5 mm posterior to the crush from wt (I) and Bcl-2tg (J) mice treated with astrotoxin. Asterisks indicate crush sites; white lines mark GFAP-negative area. Bar, 1 μm. (K) Quantification of regenerating axons from optic nerve sections. Values are mean±s.e.m. (***P<0.001).

Fig. 6

Robust optic nerve regeneration and target innervation in Bcl-2tgGFAP^−/−Vim^−/− mice after P5. (A-D) Photomicrograph montages of adjacent longitudinal optic nerve sections on day 4 after optic nerve crush in P5 GFAP^−/−Vim^−/− (G/V) (A,B) and Bcl-2tgGFAP^−/−Vim^−/− (Bcl-2tg/G/V) (C,D) mice. The asterisk indicates the crush site. Bar, 100 μm. (E-G) Higher-power views of axon and growth cone morphology (40×) revealed by anti-NF staining. Bars, 40 μm (E); 20 μm (F,G); 5 μm (H-K). Epifluorescence photomicrographs of coronal brain sections from GFAP^−/−Vim^−/− (H,J) and Bcl-2tgGFAP^−/−Vim^−/− (I,K) mice subjected to optic nerve crush on P14 and examined 4 days later. Note the positive labeling in the ipsilateral optic chiasm (K), dLGN and vLGN, and the optic tract (arrowheads) (I) of Bcl-2tgGFAP^−/−Vim^−/− brain sections. Dotted lines outline the dLGN. Bar, 200 μm.

Fig. 7

Target innervation by regenerating axons of Bcl-2tgGFAP^−/−Vim^−/− mice after P5. Photomicrographs of whole-mount retinas from Bcl-2tg (A), GFAP^−/−Vim^−/− (B), and Bcl-2tgGFAP^−/−Vim^−/− (C) mice on day 11 after optic nerve injury, showing FluoroGold-labeled RGCs. Bar, 50 μm. (D) Number of retrogradely labeled RGCs in retinal whole-mounts. Values are mean±s.e.m. (***P<0.001).