First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01658
Journal of Cell Science 118, 863-872 (2005)
Published by The Company of Biologists 2005
Re-establishing the regenerative potential of central nervous system axons in postnatal mice
Kin-Sang Cho1,
Liu Yang1,
Bin Lu1,*,
Hong Feng Ma1,
Xizhong Huang1,
Milos Pekny2,
and
Dong Feng Chen1,
1 Schepens Eye Research Institute, Program in Neuroscience and Department of Ophthalmology, Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA
2 The Arvid Carlsson Institute for Neuroscience, Institute of Clinical Neuroscience, Sahlgrenska Academy, Göteborg University, Medicinaregatan 9A, SE-413 90 Göteborg, Sweden
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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 higher-magnification views of axon and growth cone morphologies (100x). Asterisks indicates the crush site; arrowheads indicate growth-cone-like structures. Bar, 100 µm (A-C); 40 µm (D,E); 10 µm (F,G).
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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.
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Fig. 3. Quantitative assessment of axonal regeneration. (A-D) Photomicrographs of FluoroGold-labeled RGCs in whole-mount 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.
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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).
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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).
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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 (40x) 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.
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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).
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© The Company of Biologists Ltd 2005
