Stimulation of neural regeneration in the mouse retina

Abstract

Müller glia can serve as a source of new neurons after retinal damage in both fish and birds. Investigations of regeneration in the mammalian retina in vitro have provided some evidence that Müller glia can proliferate after retinal damage and generate new rods; however, the evidence that this occurs in vivo is not conclusive. We have investigated whether Müller glia have the potential to generate neurons in the mouse retina in vivo by eliminating ganglion and amacrine cells with intraocular NMDA injections and stimulating Müller glial to re-enter the mitotic cycle by treatment with specific growth factors. The proliferating Müller glia dedifferentiate and a subset of these cells differentiated into amacrine cells, as defined by the expression of amacrine cell-specific markers Calretinin, NeuN, Prox1, and GAD67-GFP. These results show for the first time that the mammalian retina has the potential to regenerate inner retinal neurons in vivo.

Fig. 1.

Müller glia proliferation in adult mouse retina. (A) Treatment scheme: NMDA induced damage at day 0 (D0) with subsequent EGF injection at day 2 (D2). BrdU was applied at D2 to identify cell proliferation. (B–F) NMDA damage followed by EGF treatment induces significant proliferation (C, D, F), while EGF (B) or NMDA neurotoxic damage (E) alone do not (E-F, BrdU = black pseudosquares). RGCs are shown in retinal flatmounts of adult mice labeled for neurofilament M (NFM, red) without (B) and after cell loss due to NMDA neurotoxic damage (C and D). (E) NMDA alone produces some proliferating cells around the optic nerve head and occasional BrdU+ cells appear at the very periphery. NMDA damage followed by EGF (F) results in significant proliferation and correlates with the amount NMDA given (C and D). (G) There are only slightly more BrdU labeled cells in the central retina compared with the peripheral retina after NMDA damage and EGF treatment (see also Fig. S2A). (H) The number of BrdU+ cells in NMDA-damaged retinas decreases with time after treatment (see also Fig. S2 B–D). Images show single confocal planes of 2 μm (B-D red channel) and z axis projections of 70 × 1 μm (B-D green channel). (Scale bars: 10 μm (B–D)).

Fig. 2.

Müller glia proliferation is inducible after retinal damage. Sox2 (A) and Sox9 (B) label Müller glia, as shown by colabeling with CRALBP. After NMDA damage, GFAP-Cre::EYFP labels Müller glia, as shown by double-labeling with Sox9 (C) and CRALBP (D, D′,D″). (E) Confocal image of retina flatmounts show BrdU+/Sox2+ double-labeled cells at depths corresponding to Müller glia as early as 4 h after induction of proliferation by various factors following NMDA damage and EGF. The xz- (E′) and yz-views (E″) represent single optical sections through the indicated area (crosshair). (F) After NMDA damage followed by 4 consecutive injections of FGF1/insulin, Müller glia up-regulate PCNA and treatment (F), BrdU (G and H), Ki67 (I) and phospho-histone3 (J). (K) GFAP-Cre::EYFP Müller glia express also express Pax6. Interestingly, at this time point (D6 after damage and treatment) some GFAP-Cre::EYFP Müller glia can be found in the ONL (C and D). (Scale bars: 10 μm.)

Fig. 3.

BrdU+ cells express Pax6, NeuN and Calretinin (CalR) and GAD67-GFP. (A–C) After NMDA induced retinal damage and EGF or FGF1 treatment, we found BrdU+ neurons labeled with Pax6 (A), NeuN (B), and Calretinin (C) 8 to 14 days later. Images show single focal planes of 2 μm. (Scale bars: 5 μm in orthogonal and inset views; 10 μm all other views.) GAD67-GFP mice express GFP in immature neurons, a subset of mature RGCs, horizontal and all GABAergic amacrine cells in adult mouse retina. NMDA/4xFGF1+insulin treatment of GAD67-GFP mouse retinas leads to BrdU+ cells in both the ganglion cell layer (F) and the amacrine cell layer (D, E, G and H). (H) At D30 some BrdU+ GAD67-GFP+ cells are also labeled for Prox1. (I) Graphs showing the number of BrdU+/GAD67-GFP+ cells as a function of days after NMDA damage. (J) Most of these cells are located in the inner nuclear layer, but some cells are found in the ganglion cell and horizontal cell layers. Images show z axis projections of 20 × 0.5 μm, i.e., 20 planes, 0.5 μm apart (D), 1 × 0.5 μm (d), 3 × 0.5 μm (E, e), 16 × 0.25 μm (F, f), 2 × 0.25 μm (G, g), 2 × 1 μm (H), and 1 × 1 μm (h). (Scale bars: 5 μm in orthogonal and inset views; 10 μm all other views.)