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Generation of immature retinal neurons from proliferating cells in the pars plana after retinal histogenesis in mice with retinal degeneration.

Nishiguchi KM, Kaneko H, Nakamura M, Kachi S, Terasaki H - Mol. Vis. (2009)

Bottom Line: Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically.Tracking experiments using BrdU indicated that some of recoverin-positive cells in the pars plana (approximately 3%) were generated after retinal histogenesis, and few were produced at or after postnatal day 24 (P24).Moreover, some (approximately 1.5%) of the recoverin-positive cells were newly generated from dividing retinal progenitors in the adult pars plana.

View Article: PubMed Central - PubMed

Affiliation: Nagoya University Graduate School of Medicine, Department of Ophthalmology, Nagoya, Japan. kojinish@med.nagoya-u.ac.jp

ABSTRACT

Purpose: To study the differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium after retinal histogenesis in mice with inherited or acquired retinal degeneration.

Methods: Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically. The distribution and differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium of mice with inherited (C3H/HeJ) and acquired (C57BL mice injected with 60 mg/kg N-methyl-N-nitrosourea) retinal degeneration were assessed. Proliferating retinal progenitors were labeled with bromodeoxyuridine (BrdU), and they were studied histologically using retinal markers.

Results: Many cells of rod and cone photoreceptor lineage were identified within the ciliary epithelium of the pars plana in adult mice with inherited retinal degeneration. Tracking experiments using BrdU indicated that some of recoverin-positive cells in the pars plana (approximately 3%) were generated after retinal histogenesis, and few were produced at or after postnatal day 24 (P24). The induction of acquired retinal degeneration in adult wild-type mice (P30) increased the number of BrdU-positve cells by roughly fourfold and recoverin-positive cells by approximately 17-fold in the pars plana. Moreover, some (approximately 1.5%) of the recoverin-positive cells were newly generated from dividing retinal progenitors in the adult pars plana.

Conclusions: In response to retinal damage, an increased number of immature retinal neurons/retinal precursors was observed in the pars plana of mice with acquired and inherited retinal degeneration. Some of these cells differentiated from proliferating cells even after retinal histogenesis.

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Generation of recoverin-positive cells in the pars plana after retinal histogenesis in rd1 mice. Double-headed arrows indicate the pars plana. A: Cells positive for both BrdU (labeled at P6) and recoverin (arrowhead) were found in the P30 pars plana. Note that numerous cells in the neuroblast layer were labeled with BrdU at P6. B: Cells positive for both BrdU (labeled at P12) and recoverin (filled arrowhead) were identified in the pars plana at P30. A cell positive for BrdU but negative for recoverin is also present in the inset (open arrowhead). Only rare BrdU-positive cells, mostly blood cells (cells in the lower right corner), were identified in the retina, suggesting that gross retinal histogenesis was already complete by P12. Note that immunopositive cells in the peripheral retina showed an oblique alignment, which contrasted with those in the pars plana; the differential susceptibility of the retina and ciliary epithelium to mounting artifacts is one of the features that sometimes distinguished the cilioretinal border. C: Hematoxylin and eosin staining of eye section is from a P12 rd1 mouse. The box roughly corresponds to the area from which images A and B were obtained. D: Cells positive for both BrdU (labeled at P18) and recoverin (arrowhead) were identified in the P30 pars plana. E,F: The proportion (%) of cells positive for BrdU (injected at P6, P12, P18, and P24) among recoverin-positive cells in the P30 pars plana (E) and similarly in the pars plana of mice (enucleated at P9, P12, P20, and P30) after BrdU injection at P6 (F) are presented. After the number of recoverin-positive cells within 320-µm width of the pars plan were determined from a single optical scan (10.0 μm thick), the number of those also positive for BrdU were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the proportion of BrdU-positive cells among recoverin-positive cells per animal. Average proportion (%; mean±SEM) for each category were determined from the following number of animals. The numbers of mice used in each experiment is summarized in Table 1. A dose of BrdU injected was 50 mg/kg. A and B are thick scans each merged from 3 scans (each scan was 10.0 μm thick) while D is also a thick scan but merged image from 2 scans (each scan was 3.9 μm thick). Scale bar equals 25 μm (D) and 50 μm in A, B, and C. Abbreviation: recoverin (Rcv).
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f6: Generation of recoverin-positive cells in the pars plana after retinal histogenesis in rd1 mice. Double-headed arrows indicate the pars plana. A: Cells positive for both BrdU (labeled at P6) and recoverin (arrowhead) were found in the P30 pars plana. Note that numerous cells in the neuroblast layer were labeled with BrdU at P6. B: Cells positive for both BrdU (labeled at P12) and recoverin (filled arrowhead) were identified in the pars plana at P30. A cell positive for BrdU but negative for recoverin is also present in the inset (open arrowhead). Only rare BrdU-positive cells, mostly blood cells (cells in the lower right corner), were identified in the retina, suggesting that gross retinal histogenesis was already complete by P12. Note that immunopositive cells in the peripheral retina showed an oblique alignment, which contrasted with those in the pars plana; the differential susceptibility of the retina and ciliary epithelium to mounting artifacts is one of the features that sometimes distinguished the cilioretinal border. C: Hematoxylin and eosin staining of eye section is from a P12 rd1 mouse. The box roughly corresponds to the area from which images A and B were obtained. D: Cells positive for both BrdU (labeled at P18) and recoverin (arrowhead) were identified in the P30 pars plana. E,F: The proportion (%) of cells positive for BrdU (injected at P6, P12, P18, and P24) among recoverin-positive cells in the P30 pars plana (E) and similarly in the pars plana of mice (enucleated at P9, P12, P20, and P30) after BrdU injection at P6 (F) are presented. After the number of recoverin-positive cells within 320-µm width of the pars plan were determined from a single optical scan (10.0 μm thick), the number of those also positive for BrdU were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the proportion of BrdU-positive cells among recoverin-positive cells per animal. Average proportion (%; mean±SEM) for each category were determined from the following number of animals. The numbers of mice used in each experiment is summarized in Table 1. A dose of BrdU injected was 50 mg/kg. A and B are thick scans each merged from 3 scans (each scan was 10.0 μm thick) while D is also a thick scan but merged image from 2 scans (each scan was 3.9 μm thick). Scale bar equals 25 μm (D) and 50 μm in A, B, and C. Abbreviation: recoverin (Rcv).

Mentions: All experimental procedures were performed in accordance with the guidelines of the Institute for Laboratory Animal Research (Guide for the Care and Use of Laboratory Animals) and Nagoya University School of Medicine for the use of animals. C57BL/6J mice were used as wild-type controls and for the N-methyl-N-nitrosourea (MNU; Clea, Tokyo, Japan) injection study. C3H/HeJ mice (rd1 mice; Clea, Tokyo, Japan) were used as a murine model of inherited retinal degeneration [20]. All mice were fed on a basal diet (CE-2; Clea, Tokyo, Japan) and water. They were kept on a 12-h light-dark cycle in a standard cage placed in a temperature- and humidity-controlled environment. The number of mice used in each quantitative experiment is indicated in Table 1. A total estimate of at least 200 mice was used throughout the study (128 mice were used for quantification purpose).


Generation of immature retinal neurons from proliferating cells in the pars plana after retinal histogenesis in mice with retinal degeneration.

Nishiguchi KM, Kaneko H, Nakamura M, Kachi S, Terasaki H - Mol. Vis. (2009)

Generation of recoverin-positive cells in the pars plana after retinal histogenesis in rd1 mice. Double-headed arrows indicate the pars plana. A: Cells positive for both BrdU (labeled at P6) and recoverin (arrowhead) were found in the P30 pars plana. Note that numerous cells in the neuroblast layer were labeled with BrdU at P6. B: Cells positive for both BrdU (labeled at P12) and recoverin (filled arrowhead) were identified in the pars plana at P30. A cell positive for BrdU but negative for recoverin is also present in the inset (open arrowhead). Only rare BrdU-positive cells, mostly blood cells (cells in the lower right corner), were identified in the retina, suggesting that gross retinal histogenesis was already complete by P12. Note that immunopositive cells in the peripheral retina showed an oblique alignment, which contrasted with those in the pars plana; the differential susceptibility of the retina and ciliary epithelium to mounting artifacts is one of the features that sometimes distinguished the cilioretinal border. C: Hematoxylin and eosin staining of eye section is from a P12 rd1 mouse. The box roughly corresponds to the area from which images A and B were obtained. D: Cells positive for both BrdU (labeled at P18) and recoverin (arrowhead) were identified in the P30 pars plana. E,F: The proportion (%) of cells positive for BrdU (injected at P6, P12, P18, and P24) among recoverin-positive cells in the P30 pars plana (E) and similarly in the pars plana of mice (enucleated at P9, P12, P20, and P30) after BrdU injection at P6 (F) are presented. After the number of recoverin-positive cells within 320-µm width of the pars plan were determined from a single optical scan (10.0 μm thick), the number of those also positive for BrdU were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the proportion of BrdU-positive cells among recoverin-positive cells per animal. Average proportion (%; mean±SEM) for each category were determined from the following number of animals. The numbers of mice used in each experiment is summarized in Table 1. A dose of BrdU injected was 50 mg/kg. A and B are thick scans each merged from 3 scans (each scan was 10.0 μm thick) while D is also a thick scan but merged image from 2 scans (each scan was 3.9 μm thick). Scale bar equals 25 μm (D) and 50 μm in A, B, and C. Abbreviation: recoverin (Rcv).
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Related In: Results  -  Collection

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f6: Generation of recoverin-positive cells in the pars plana after retinal histogenesis in rd1 mice. Double-headed arrows indicate the pars plana. A: Cells positive for both BrdU (labeled at P6) and recoverin (arrowhead) were found in the P30 pars plana. Note that numerous cells in the neuroblast layer were labeled with BrdU at P6. B: Cells positive for both BrdU (labeled at P12) and recoverin (filled arrowhead) were identified in the pars plana at P30. A cell positive for BrdU but negative for recoverin is also present in the inset (open arrowhead). Only rare BrdU-positive cells, mostly blood cells (cells in the lower right corner), were identified in the retina, suggesting that gross retinal histogenesis was already complete by P12. Note that immunopositive cells in the peripheral retina showed an oblique alignment, which contrasted with those in the pars plana; the differential susceptibility of the retina and ciliary epithelium to mounting artifacts is one of the features that sometimes distinguished the cilioretinal border. C: Hematoxylin and eosin staining of eye section is from a P12 rd1 mouse. The box roughly corresponds to the area from which images A and B were obtained. D: Cells positive for both BrdU (labeled at P18) and recoverin (arrowhead) were identified in the P30 pars plana. E,F: The proportion (%) of cells positive for BrdU (injected at P6, P12, P18, and P24) among recoverin-positive cells in the P30 pars plana (E) and similarly in the pars plana of mice (enucleated at P9, P12, P20, and P30) after BrdU injection at P6 (F) are presented. After the number of recoverin-positive cells within 320-µm width of the pars plan were determined from a single optical scan (10.0 μm thick), the number of those also positive for BrdU were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the proportion of BrdU-positive cells among recoverin-positive cells per animal. Average proportion (%; mean±SEM) for each category were determined from the following number of animals. The numbers of mice used in each experiment is summarized in Table 1. A dose of BrdU injected was 50 mg/kg. A and B are thick scans each merged from 3 scans (each scan was 10.0 μm thick) while D is also a thick scan but merged image from 2 scans (each scan was 3.9 μm thick). Scale bar equals 25 μm (D) and 50 μm in A, B, and C. Abbreviation: recoverin (Rcv).
Mentions: All experimental procedures were performed in accordance with the guidelines of the Institute for Laboratory Animal Research (Guide for the Care and Use of Laboratory Animals) and Nagoya University School of Medicine for the use of animals. C57BL/6J mice were used as wild-type controls and for the N-methyl-N-nitrosourea (MNU; Clea, Tokyo, Japan) injection study. C3H/HeJ mice (rd1 mice; Clea, Tokyo, Japan) were used as a murine model of inherited retinal degeneration [20]. All mice were fed on a basal diet (CE-2; Clea, Tokyo, Japan) and water. They were kept on a 12-h light-dark cycle in a standard cage placed in a temperature- and humidity-controlled environment. The number of mice used in each quantitative experiment is indicated in Table 1. A total estimate of at least 200 mice was used throughout the study (128 mice were used for quantification purpose).

Bottom Line: Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically.Tracking experiments using BrdU indicated that some of recoverin-positive cells in the pars plana (approximately 3%) were generated after retinal histogenesis, and few were produced at or after postnatal day 24 (P24).Moreover, some (approximately 1.5%) of the recoverin-positive cells were newly generated from dividing retinal progenitors in the adult pars plana.

View Article: PubMed Central - PubMed

Affiliation: Nagoya University Graduate School of Medicine, Department of Ophthalmology, Nagoya, Japan. kojinish@med.nagoya-u.ac.jp

ABSTRACT

Purpose: To study the differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium after retinal histogenesis in mice with inherited or acquired retinal degeneration.

Methods: Immunoreactivity to anti-recoverin, rhodopsin, and Pax6 antibodies and binding to peanut agglutinin were analyzed histologically. The distribution and differentiation of immature retinal neurons/retinal precursors in the ciliary epithelium of mice with inherited (C3H/HeJ) and acquired (C57BL mice injected with 60 mg/kg N-methyl-N-nitrosourea) retinal degeneration were assessed. Proliferating retinal progenitors were labeled with bromodeoxyuridine (BrdU), and they were studied histologically using retinal markers.

Results: Many cells of rod and cone photoreceptor lineage were identified within the ciliary epithelium of the pars plana in adult mice with inherited retinal degeneration. Tracking experiments using BrdU indicated that some of recoverin-positive cells in the pars plana (approximately 3%) were generated after retinal histogenesis, and few were produced at or after postnatal day 24 (P24). The induction of acquired retinal degeneration in adult wild-type mice (P30) increased the number of BrdU-positve cells by roughly fourfold and recoverin-positive cells by approximately 17-fold in the pars plana. Moreover, some (approximately 1.5%) of the recoverin-positive cells were newly generated from dividing retinal progenitors in the adult pars plana.

Conclusions: In response to retinal damage, an increased number of immature retinal neurons/retinal precursors was observed in the pars plana of mice with acquired and inherited retinal degeneration. Some of these cells differentiated from proliferating cells even after retinal histogenesis.

Show MeSH
Related in: MedlinePlus