J Korean Ophthalmol Soc.  2013 Jun;54(6):945-953. 10.3341/jkos.2013.54.6.945.

Expression Profiles of F4/80 and Nestin in Ocular Immune Cells Following Pharmaceutically Induced Retinal Degeneration in Adult Mice

• Ahn SI
• Ohn YH
• Park TK

• KMID: 2217272
• DOI: http://doi.org/10.3341/jkos.2013.54.6.945

Abstract

PURPOSE
To evaluate the expression patterns of F4/80 and nestin in the ciliary body and the optic nerve following N-methyl-N-nitrosourea (NMU)-induced retinal degeneration in adult mice.
METHODS
After intraperitoneal injection of MNU (60 mg/kg) in adult mice, the eyes were enucleated at 2, 4, 7 and 30 days. Hematoxylin and eosin (H&E) stain, terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stain and immunohistochemical stains of F/80 and nestin were performed.
RESULTS
After MNU treatment, the photoreceptors were destroyed by cell apoptosis. According to immunohistochemistry, F4/80 and nestin were not co-expressed in the control group, but F4/80 was expressed within the ciliary body and optic nerve in the MNU-treated group; the expression of nestin also increased. In the outer nuclear layer, F4/80 and nestin co-expressing cells were observed.
CONCLUSIONS
In response to retinal damage, the F4/80 and nestin co-expressing cells migrated to the retina from the ciliary body and optic nerve and were activated.

Keyword

F4/80; MNU; Nestin; Retinal degeneration

MeSH Terms

Adult
Animals
Apoptosis
Ciliary Body
Coloring Agents
Eosine Yellowish-(YS)
Eye
Hematoxylin
Humans
Immunohistochemistry
Injections, Intraperitoneal
Intermediate Filament Proteins
Methylnitrosourea
Mice
Nerve Tissue Proteins
Optic Nerve
Retina
Retinal Degeneration
Retinaldehyde
Coloring Agents
Eosine Yellowish-(YS)
Hematoxylin
Intermediate Filament Proteins
Methylnitrosourea
Nerve Tissue Proteins
Retinaldehyde

Figure

• 

  Figure 1. H&E staining results of the retina. (A) Normal. (B) 2 days after MNU treatment, the retinal thickness is slightly decreased and the arrangement of PRL and ONL is distorted. (C) 7 days after MNU treatment, the depletion of PRL and RPE are noticed and the ONL is reduced to a few layers of cells. (D) 30 days after MNU treatment, the ONL completely disappeared (RPE = retinal pigment epithelium; PRL = photoreceptor layer; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer).

• 

  Figure 2. TUNEL staining results of the retina. (A) No TUNEL+ cells are observed in the control group. (B) Large number of TUNEL+ cells are shown in the ONL at 2 days after MNU treatment. (C) Seven days after MNU treatment, small num-ber of TUNEL+ cells are remained in the ONL and GCL. (D) Thirty days after MNU treatment, no TUNEL+ cells are observed as the ONL disappeared (RPE = retinal pigment epithelium; PRL = photoreceptor layer; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer).

• 

  Figure 3. Immunochemistry examination of the optic nerve head. Immunofluorescent labeling with F4/80 (red) and nestin (green). In the control optic nerve head, there are some of F4/80+ cells (A: arrowhead) and nestin+ cells (B: asterisk), but no cells co-ex-pressed F4/80 and nestin (C: merged image of A and B). Two days after MNU treatment, some of F4/80+ cells are also positive for nestin (D-F: arrow). Four days after MNU treatment, large number of F4/80 and nestin co-expressing cells are observed (G-I: arrow). Seven days after MNU treatment, these cells are decreased (J-L). Scale bar: 100 μ m.

• 

  Figure 4. Immunochemistry examination of the ciliary body. Immunofluorescent labeling with F4/80 (red) and nestin (green). In the control ciliary body, F4/80 immunoactivity is seen in the ciliary epithelium, but nestin is not expressed (A-C: arrow). The stroma of ciliary body is non-specifically stained by nestin, but F4/80 is not expressed (B, E, H: asterisk). In the choroid, F4/80 and nestin co-expressing cells are observed (A-C: arrowhead). Two days after MNU treatment, F4/80 immunoactivity of ciliary epithelium is more increased (D: arrow) and there are some of cells which co-expressed F4/80 and nestin (D-F: arrowhead). Four days after MNU treatment, F4/80+ cells are seen in the ciliary epithelium (G: arrow), and F4/80 and nestin co-expressing cells are seen in the fibrovascular tissue of ciliray body (G-L: arrowhead). (CB: ciliary body). Scale bar: 100 μ m.

• 

  Figure 5. Immunochemistry examination of the retina. Immunofluorescent labeling with F4/80 (red) and nestin (green). In the con-trol retina, there are no F4/80+ cells (A), but nestin is found in retinal blood vessels (B: arrowheads) and muller cells (B: asterisk). Two days after MNU treatment, F4/80 and nestin are co-expressed between INL and ONL (D-F: arrow). Four days after MNU treatment, F4/80 is also expressed in the ONL (G-I: arrow). Nestin immunoactivity of the muller cells was increased (E, H) (PRL = photoreceptor layer; ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexi-form layer; GCL = ganglion cell layer). Scale bar: 50 μ m.

Reference

References

1. Herrold KM. Pigmentary degeneration of the retina induced by N-methyl-N-nitrosourea. An experimental study in syrian hamsters. Arch Ophthalmol. 1967; 78:650–3.

2. Nakajima M, Nambu H, Shikata N. . Pigmentary degeneration induced by N-methyl-N-nitrosourea and the fate of pigment epi-thelial cells in the rat retina. Pathol Int. 1996; 46:874–82.

3. Nambu H, Yuge K, Nakajima M. . Morphologic characteristics of N-methyl-N-nitrosourea-induced retinal degeneration in C57BL mice. Pathol Int. 1997; 47:377–83.

4. Nakajima M, Yuge K, Senzaki H. . Photoreceptor apoptosis in-duced by a single systemic administration of N-methyl-N-nitro-sourea in the rat retina. Am J Pathol. 1996; 148:631–41.

5. Swirski FK, Nahrendorf M, Etzrodt M. . Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science. 2009; 325:612–6.

6. Becher B, Prat A, Antel JP. Brain-immune connection: immuno-reg-ulatory properties of CNS-resident cells. Glia. 2000; 29:293–304.

7. Langmann T. Microglia activation in retinal degeneration. J Leukoc Biol. 2007; 81:1345–51.

8. Chen L, Yang P, Kijlstra A. Distribution, markers, and functions of retinal microglia. Ocul Immunol Inflamm. 2002; 10:27–39.

9. Streit WJ. Microglia as neuroprotective, immunocompetent cells of the CNS. Glia. 2002; 40:133–9.

10. Provis JM, Diaz CM, Penfold PL. Microglia in human retina: a het-erogeneous population with distinct ontogenies. Perspect Dev Neurobiol. 1996; 3:213–22.

11. Hume DA, Perry VH, Gordon S. Immunohistochemical local-ization of a macrophage-specific antigen in developing mouse reti-na: phagocytosis of dying neurons and differentiation of microglial cells to form a regular array in the plexiform layers. J Cell Biol. 1983; 97:253–7.

12. Haidl ID, Jefferies WA. The macrophage cell surface glycoprotein F4/80 is a highly glycosylated proteoglycan. Eur J Immunol. 1996; 26:1139–46.

13. Hirsch S, Austyn JM, Gordon S. Expression of the macro-phage-specific antigen F4/80 during differentiation of mouse bone marrow cells in culture. J Exp Med. 1981; 154:713–25.

14. Austyn JM, Gordon S. F4/80, a monoclonal antibody directed spe-cifically against the mouse macrophage. Eur J Immunol. 1981; 11:805–15.

15. Perry VH, Hume DA, Gordon S. Immunohistochemical local-ization of macrophages and microglia in the adult and developing mouse brain. Neuroscience. 1985; 15:313–26.

16. McMenamin PG. Dendritic cells and macrophages in the uveal tract of the normal mouse eye. Br J Ophthalmol. 1999; 83:598–604.

17. McMenamin PG, Crewe J, Morrison S, Holt PG. Immunomorphologic studies of macrophages and MHC class II-positive dendritic cells in the iris and ciliary body of the rat, mouse, and human eye. Invest Ophthalmol Vis Sci. 1994; 35:3234–50.

18. Kezic J, McMenamin PG. Differential turnover rates of mono-cyte-derived cells in varied ocular tissue microenvironments. J Leukoc Biol. 2008; 84:721–9.

19. Kaneko H, Nishiguchi KM, Nakamura M. . Characteristics of bone marrow-derived microglia in the normal and injured retina. Invest Ophthalmol Vis Sci. 2008; 49:4162–8.

20. Xu H, Sta Iglesia DD, Kielczewski JL. . Characteristics of pro-genitor cells derived from adult ciliary body in mouse, rat, and hu-man eyes. Invest Ophthalmol Vis Sci. 2007; 48:1674–82.

21. Michalczyk K, Ziman M. Nestin structure and predicted function in cellular cytoskeletal organisation. Histol Histopathol. 2005; 20:665–71.

22. Kohno H, Sakai T, Kitahara K. Induction of nestin, Ki-67, and cy-clin D1 expression in Muller cells after laser injury in adult rat retina. Graefes Arch Clin Exp Ophthalmol. 2006; 244:90–5.

23. Xue L, Ding P, Xiao L. . Nestin, a new marker, expressed in Muller cells following retinal injury. Can J Neurol Sci. 2010; 37:643–9.

24. Sahin Kaya S, Mahmood A, Li Y. . Expression of nestin after traumatic brain injury in rat brain. Brain Res. 1999; 840:153–7.

25. Wohl SG, Schmeer CW, Witte OW, Isenmann S. Proliferative re-sponse of microglia and macrophages in the adult mouse eye after optic nerve lesion. Invest Ophthalmol Vis Sci. 2010; 51:2686–96.

26. Warzok R, Thust R, Schneider J. . [Induction of malformations by N-methyl-N-nitrosourea (MNU) (author's transl))]. Exp Pathol (Jena). 1977; 13:11–9.

27. Bond SL, Singh SM. Methyl nitrosourea induced unscheduled DNA synthesis in vivo in mice. Effects of background genotype on excision repair during aging. Mech Ageing Dev. 1987; 41:177–87.

28. Alliot F, Rutin J, Leenen PJ, Pessac B. Pericytes and peri-endothelial cells of brain parenchyma vessels co-express amino-peptidase N, aminopeptidase A, and nestin. J Neurosci Res. 1999; 58:367–78.

29. Zhang C, Lam TT, Tso MO. Heterogeneous populations of micro-glia/macrophages in the retina and their activation after retinal is-chemia and reperfusion injury. Exp Eye Res. 2005; 81:700–9.

30. Wohl SG, Schmeer CW, Friese T. . In situ dividing and phag-ocytosing retinal microglia express nestin, vimentin, and NG2 in vivo. PloS One. 2011; 6:e22408.

31. Ogino H, Ito M, Matsumoto K. . Retinal degeneration induced by N-methyl-N-nitrosourea and detection of 7-methyldeox-yguanosine in the rat retina. Toxicol Pathol. 1993; 21:21–5.

32. Nickerson PE, Emsley JG, Myers T, Clarke DB. Proliferation and expression of progenitor and mature retinal phenotypes in the adult mammalian ciliary body after retinal ganglion cell injury. Invest Ophthalmol Vis Sci. 2007; 48:5266–75.

33. Lijinsky W, Kovatch RM, Saavedra JE. Carcinogenesis and muta-genesis by N-nitroso compounds having a basic center. Cancer Lett. 1992; 63:101–7.

34. Yoshizawa K, Tsubura A. [Characteristics of N-methyl-N-nitro-sourea-induced retinal degeneration in animals and application for the therapy of human retinitis pigmentosa]. Nippon Ganka Gakkai zasshi. 2005; 109:327–37.

35. Yoshizawa K, Nambu H, Yang J. . Mechanisms of photo-receptor cell apoptosis induced by N-methyl-N-nitrosourea in Sprague-Dawley rats. Lab Invest. 1999; 79:1359–67.

36. Yang J, Lin S, Hu S. . [The toxic effect of N-methyl-N- nitro-sourea on retina in rats]. Yan ke xue bao. 2004; 20:249–54.

37. Jeong E, Paik SS, Jung SW. . Morphological and functional evaluation of an animal model for the retinal degeneration induced by N-methyl-N-nitrosourea. Anat Cell Biol. 2011; 44:314–23.

38. Thanos S. Sick photoreceptors attract activated microglia from the ganglion cell layer: a model to study the inflammatory cascades in rats with inherited retinal dystrophy. Brain Res. 1992; 588:21–8.

39. Roque RS, Imperial CJ, Caldwell RB. Microglial cells invade the outer retina as photoreceptors degenerate in Royal College of Surgeons rats. Invest Ophthalmol Vis Sci. 1996; 37:196–203.

40. Caicedo A, Espinosa-Heidmann DG, Pina Y. . Blood-derived macrophages infiltrate the retina and activate Muller glial cells un-der experimental choroidal neovascularization. Exp Eye Res. 2005; 81:38–47.

41. Kawaguchi A, Miyata T, Sawamoto K. . Nestin-EGFP trans-genic mice: visualization of the self-renewal and multipotency of CNS stem cells. Mol Cell Neurosci. 2001; 17:259–73.

42. Walcott JC, Provis JM. Muller cells express the neuronal progeni-tor cell marker nestin in both differentiated and undifferentiated human foetal retina. Clin Experiment Ophthalmol. 2003; 31:246–9.

43. Xue LP, Lu J, Cao Q. . Nestin expression in Muller glial cells in postnatal rat retina and its upregulation following optic nerve transection. Neuroscience. 2006; 143:117–27.

44. Xue LP, Lu J, Cao Q. . Muller glial cells express nestin coupled with glial fibrillary acidic protein in experimentally induced glau-coma in the rat retina. Neuroscience. 2006; 139:723–32.