Figure 1.
A: preparation of distal optic nerve (ON) lesion; retinal ganglion cell (RGC) axons were intradurally transected approx. 2 mm behind the eyeball. B: immediately after surgery, rats received intraperitoneal injections of BrdU twice daily up to 3 or 7 days. Animals were sacrificed at days 3, 7, and 14 or 8 weeks after ON axotomy (indicated by an X in the time axis; for details, see Materials and Methods). Cell fate analyses were performed 14 days and 8 weeks after injury. Each group consisted of 7 rats. C: Every 25th horizontal cross section of the eye cup including the neural retina was placed on consecutive slides (24 slides in sum, 10 sections per slide) resulting in a representative coverage of the whole retina on one slide.
Table 1.
Primary antibodies employed.
Figure 2.
Nestin+ microglia in the naïve retina.
Immunofluorescent labeling with Iba1 (red) and nestin antisera (green) as well as DAPI nuclear staining (blue). Resting microglia, mainly found in the GCL and IPL, had fine branched processes which expressed nestin in some, but not all processes (A,B arrowheads; boxes 1 to 3 in B, higher magnification in C-C″, D-D″ and E-E′, respectively). Ea–f and E′a–f represent the gallery of 1 µm optical sections of this z-stack. Nestin was also found in retinal blood vessels (arrows) and in few radial macroglial processes (asterisks). The micrographs in A–D are merged z-stacked images of 1 µm optical sections to illustrate the entire cell dimension. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer. Scale bar (A,B) 50 µm, (C-C″,D-D″, E,E′) 20 µm.
Figure 3.
Retinal nestin+ microglia after ON axotomy.
A–I: Immunofluorescent labeling with Iba1 (red), nestin (green) and DAPI (blue). 3 days after ON axotomy, rather rounded nestin+ microglia were found in the GCL and IPL (A, arrowhead-marked cell in B). C–I: 7 days after ON lesion, a significant increase in nestin immunoreactivity was observed predominantly in the processes of astrocytes and Müller glia (C, examples are illustrated with asterisks). Increased numbers of Iba1+ microglia were mainly found in the inner retinal layers and the OPL, where the majority express nestin (arrowheads). A few ameboid Iba1+ cells observed in the photoreceptor layer (arrows) were also nestin+ (o). Some retinal microglia lacked nestin filaments (D, box 1, as ortho view in E, higher magnification in G-G″), however, the majority expressed nestin either perinuclearly (D, box 2, as ortho view in F, higher magnification in H-H″) or in their long processes (D, box 3, higher magnification in I-I″). J: Absolute numbers of total and nestin+ microglia 3, 7, 14 days, and 8 weeks after ON transection and in the naïve retina. 7 to 14 days after ON axotomy, the number of Iba1+ microglia was significantly increased compared to naïve controls, while the number of nestin+ microglia was already significantly increased 3 days after injury, reaching a maximum on day 7. Mean ± S.E.M., significant differences between lesion and corresponding control groups (* p<0.05, ** p<0.01), and between the lesion or control groups over time (+ p<0.05, ++ p<0.01) are indicated: grey and black symbols are used for the white and green diagrams, respectively. The micrographs in A,C,D,G–I are merged z-stacked images of 1 µm optical sections. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, axo: axotomy. Scale bar (A,C,D) 50 µm, (B,E,F,G-G″,H-H″,I-I″) 20 µm.
Figure 4.
Nestin expression in the ciliary body and choroid.
Immunofluorescent labeling with Iba1 (red), nestin (green), and DAPI (blue). A–C: In the ciliary stroma, the area below the dotted white line, Iba1+nestin− macrophages were found (A, box 1, higher magnification in B, arrowheads). However, in the ciliary epithelium, branched Iba1+ cells had nestin filaments in some of their processes (A, box 2, higher magnification in C,C′ arrows). Ameboid cells in the epithelium were nestin− (C,C′ asterisk). In the choroid, many ameboid Iba1+ macrophages (D,E arrowheads) were found within numerous nestin filament bundles (E,E′), however these cells per se were nestin−. The micrographs in the figures are merged z-stacked images of 1 µm optical sections to illustrate the entire cell dimension. ONL: outer nuclear layer, CB: ciliary body. Scale bar in (A,D-E′) 100 µm, (C-C′) 50 µm.
Figure 5.
Numbers of retinal BrdU+ microglia increase after ON axotomy.
A–E: Immunofluorescent labeling with Iba1 (blue), BrdU (red), and nestin antisera (green). Three days after ON axotomy, some microglia observed in inner retinal layers were BrdU+ (A, arrowhead) and some also nestin+ (the asterisk-marked cell in the box is shown in B,B′). After 7 days, BrdU+Iba1+ microglia (C, arrowheads) increased in number, especially in the GCL/IPL, and most of them co-expressed nestin (asterisk-marked cell in box 1 is shown D,D′; that of box 2 as ortho view in E). F: Absolute numbers of BrdU+ and nestin+BrdU+ microglia 3, 7, 14 days, and 8 weeks after ON transection and in the naïve retina. At all time points analyzed, numbers of BrdU+ and nestin+BrdU+ microglia were increased after ON axotomy compared to corresponding controls, reaching maximum numbers 7–14 days after injury. Mean ± S.E.M., significant differences between lesion and corresponding control groups (* p<0.05, ** p<0.01), and between the lesion or control groups over time (+ p<0.05, ++ p<0.01) are indicated: grey and black symbols are used for the white and red diagrams, respectively. The micrographs in A-D′ are merged z-stacked images of 1 µm optical sections to illustrate the entire cell dimension. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer nuclear layer, axo: axotomy. Scale bar (A,C) 50 µm, (B,B′,D,D′) 20 µm, (E) 10 µm.
Figure 6.
Retinal nestin+ microglia proliferated in situ in response to ON axotomy.
A–E: Immunofluorescent labeling with Ki67 (red), tomato lectin (A–C green; D,E blue), BrdU (A–C blue), and nestin (D,E green). A–C: within the first 2 weeks, significant numbers of Ki67+ microglia (arrows) were observed in the inner retinal layers, and Ki67 was partly co-localized with BrdU (arrowheads, co-localization is shown by asterisks). In unlesioned (D) and lesioned tissue (E), Ki67 was exclusively found in nestin+ microglia which were mainly located in the IPL. F: Absolute numbers of Ki67+ and Ki67+BrdU+ microglia 3, 7, 14 days, and 8 weeks after ON transection and in the naïve retina. Numbers of Ki67+ microglia were increased 3–14 days after ON axotomy compared to corresponding controls, reaching a maximum after 7 days. Maximum of Ki67+BrdU+ cells was also found 7 days after lesion. Mean ± S.E.M., significant differences between lesion and corresponding control groups (* p<0.05, ** p<0.01), and between the lesion or control groups over time (+ p<0.05, ++ p<0.01) are indicated: grey and black symbols are used for the white and purple diagrams, respectively. GCL: ganglion cell layer, IPL: inner plexiform layer, axo: axotomy. Scale bar in (A–C) 50 µm, (D,E) 20 µm.
Figure 7.
Percentages of the six determined microglial phenotypes after ON transection and in the naïve retina.
The nestin+ fractions are indicated by a green and nestin− fractions by a dark background. The fractions of BrdU+ microglia are illustrated as red-shaded columns. The in situ proliferating Ki67+ (always nestin+) fractions are shown as blue columns. The six resulting phenotypes were defined as follows: I) non-proliferative nestin+ microglia; II) in situ proliferating nestin+ microglia not in the S-phase of the cell cycle (BrdU−); III) in situ proliferating nestin+BrdU+; IV) nestin+BrdU+ microglia previously labeled in the S-phase (had already left the cell cycle); V) nestin−BrdU+ microglia which were degraded or had replaced their nestin filaments after cell division, and, VI) nestin−BrdU− microglia. Significant differences for a particular phenotype between the lesion and control group at a specific time point are indicated by * p<0.05, ** p<0.01.
Figure 8.
Retinal microglia express vimentin and NG2 but not GFAP.
Immunofluorescent labeling with Iba1 (A,B, A″,B″ red), vimentin (A′,A″ green), GFAP (B′,B″ green), fluorescein labeled tomato lectin (C,C″ red), NG2 (C′,C″green, D,D″, E-G,G″red), nestin (D′,D″ green), CD11b (E,F,G′,G″green), and DAPI (A-G, blue) on retinal slices. A–D: ramified retinal microglia contained vimentin filaments in their processes and within the soma, but no GFAP. Resting retinal microglia also expressed NG2 on their surface and were nestin+. E-G″: after injury, NG2+CD11b+ microglia (arrowhead), that can be clearly distinguished from NG2+CD11b− pericytes (arrows) displayed an increased NG2 immunoreactivity on their surface. The micrographs in A–E,G-G″ are merged z-stacked images of 1 µm optical sections to illustrate the entire cell dimension. GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer, OPL: outer plexiform layer, ONL: outer nuclear layer, axo: axotomy. Scale bar in (A-D″,F′-G″) 20 µm, (E) 50 µm.
Table 2.
Marker expression in retinal glia and blood vessel cells of naïve and lesioned tissue.
Figure 9.
Nestin+ and NG2+ microglia phagocytose RGCs.
Immunofluorescent labeling with Fluorogold dye (FG, A–D,H,L–N gold) as well as Brn3a (A,B magenta), Iba1 (A–F,H–J,L,N blue), nestin (E,G,I,K,N green), NG2 (F,G,J,K red), and TREM2 antisera (L–N red). 7 days after ON lesion, most of the Brn3a+ RGCs are retrogradely labeled with FG (A, arrowheads; the asterisk-marked cell in the box is shown in B as ortho view). 14 days after injury, retinal Iba1+ microglia have phagocytosed FG+ retinal ganglion cells and have incorporated the golden dye (C, box 1 is shown in higher magnification D–G, box 2 in H–K). Phagocytosing microglia were nestin+ (E,I) as well NG2+ (F,J), merged in (G,K) and expressed the TREM2 receptor on their surface (L,M, the asterisk marked cell in L,M is shown as ortho view in N). Every FG+ microglial cell was also TREM2+ (arrowheads).The micrographs in A,C,L,M are merged z-stacked images of 1 µm optical sections to illustrate entire cell dimension. GCL: ganglion cell layer, IPL: inner plexiform layer, axo: axotomy. Scale bar in (A,B,N) 20 µm, (C,L,M) 50 µm, (D–K) 10 µm.
Figure 10.
Isolated retinal microglia express nestin, vimentin and NG2.
Immunofluorescent labeling with Iba1 (A–D,D″,F-H red), nestin (A,B′,E–G,J′ green), vimentin (C,H green), tomato lectin (D′,D″,I green), NG2 (E,I,J,J′ red), and DAPI (A–D,F,G,J blue) on isolated cells after immunopanning. A–E: isolated Iba1+ microglia of naïve retinas co-expressed the intermediate filaments nestin (A,B-B″) and vimentin (C), that were observed in the processes (B,B′) as well as around the nucleus (A,C). Isolated microglia were co-labeled with tomato lectin, further confirming the microglia identity (D-D″). Immunopanning also corroborated that some of the resting retinal microglia were NG2+ and that these cells belonged to the nestin expressing microglial fraction (E). F-J′: 7 days after ON axotomy some Iba1+ microglia were nestin+ (F, arrowhead). Nestin (F,G) and vimentin filaments (H) were found around the nucleus as well as in some processes of Iba1+ retinal microglia. In addition, isolated microglia expressed NG2 after injury (I,J,J′) and these cells were also nestin+ (J′). The micrographs in B-B″,D–F,H,I are merged z-stacked images of 1 µm optical sections to illustrate entire cell dimension. In J-J′, a single micrograph from the middle of the z-stack is shown in higher magnification. Scale bar in (A,C,G) 10 µm, (B-B′,D-D″,E,F,H-J′) 20 µm.