Limits...
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.

Show MeSH

Related in: MedlinePlus

Neurogenesis in the pars plana in mice with MNU-induced photoreceptor degeneration. Double-headed arrows indicate the pars plana. MNU was injected at P30 and BrdU (50 mg/kg) was injected at P32. A, B, F: At P37, increased numbers of BrdU-positive cells were observed in the pars plana of MNU-treated mice (A) compared to untreated controls (B), which is summarized in F. Note that BrdU-positive cells in a linear alignment in the retina of B were blood cells in retinal vessels. C: Representative image of a cell positive for both recoverin and BrdU (arrowhead) in the peripheral retina at P40. D,H: Proportions of cells positive for PNA (cone) or rhodopsin (Rho; rod) among recoverin-positive cells in the pars plana (H) are presented. Note that the abrupt alteration in the cone-rod ratio signified the cilioretinal border. E, I: Rare cells positive for both recoverin and BrdU were identified in the pars plana of MNU-treated mice (E; n=8); such cells were never observed in control mice (I; n=12). G: At P37, increased numbers of recoverin-positive cells were observed in the pars plana of MNU-treated mice (n=8) as compared to the controls (n=12). For H, 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 rhodopsin and PNA were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the proportion of recoverin-positive cells that were also positive for rhodopsin (Rho) or PNA (PNA) or neither (Rcv) per animal. Average proportion (%; mean±SEM) of cells for each of the three categories were determined from 8 animals. For F, G, and I, the number of cells positive for BrdU, recoverin, or both within 320 µm width of the pars plana were determined from an optical scan (10.0 μm thick). Values from three independent images randomly obtained from a same eye were averaged. Data extracted from totals of 8 MNU-treated mice and 12 control mice were statistically processed and were presented as means±SEM A and B are thick scans merged from 2 scans (each scan was 10.0 μm thick) while C is presented as a thin scan (a single scan 3.9 μm thick). D and E are also thick scans but merged from 3 scans (each scan was 10.0 μm thick). Scale bar equals 25 μm in C-E and 50 μm in A and B. Abbreviation: recoverin (Rcv).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2629738&req=5

f8: Neurogenesis in the pars plana in mice with MNU-induced photoreceptor degeneration. Double-headed arrows indicate the pars plana. MNU was injected at P30 and BrdU (50 mg/kg) was injected at P32. A, B, F: At P37, increased numbers of BrdU-positive cells were observed in the pars plana of MNU-treated mice (A) compared to untreated controls (B), which is summarized in F. Note that BrdU-positive cells in a linear alignment in the retina of B were blood cells in retinal vessels. C: Representative image of a cell positive for both recoverin and BrdU (arrowhead) in the peripheral retina at P40. D,H: Proportions of cells positive for PNA (cone) or rhodopsin (Rho; rod) among recoverin-positive cells in the pars plana (H) are presented. Note that the abrupt alteration in the cone-rod ratio signified the cilioretinal border. E, I: Rare cells positive for both recoverin and BrdU were identified in the pars plana of MNU-treated mice (E; n=8); such cells were never observed in control mice (I; n=12). G: At P37, increased numbers of recoverin-positive cells were observed in the pars plana of MNU-treated mice (n=8) as compared to the controls (n=12). For H, 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 rhodopsin and PNA were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the proportion of recoverin-positive cells that were also positive for rhodopsin (Rho) or PNA (PNA) or neither (Rcv) per animal. Average proportion (%; mean±SEM) of cells for each of the three categories were determined from 8 animals. For F, G, and I, the number of cells positive for BrdU, recoverin, or both within 320 µm width of the pars plana were determined from an optical scan (10.0 μm thick). Values from three independent images randomly obtained from a same eye were averaged. Data extracted from totals of 8 MNU-treated mice and 12 control mice were statistically processed and were presented as means±SEM A and B are thick scans merged from 2 scans (each scan was 10.0 μm thick) while C is presented as a thin scan (a single scan 3.9 μm thick). D and E are also thick scans but merged from 3 scans (each scan was 10.0 μm thick). Scale bar equals 25 μm in C-E and 50 μm in A and B. 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)

Neurogenesis in the pars plana in mice with MNU-induced photoreceptor degeneration. Double-headed arrows indicate the pars plana. MNU was injected at P30 and BrdU (50 mg/kg) was injected at P32. A, B, F: At P37, increased numbers of BrdU-positive cells were observed in the pars plana of MNU-treated mice (A) compared to untreated controls (B), which is summarized in F. Note that BrdU-positive cells in a linear alignment in the retina of B were blood cells in retinal vessels. C: Representative image of a cell positive for both recoverin and BrdU (arrowhead) in the peripheral retina at P40. D,H: Proportions of cells positive for PNA (cone) or rhodopsin (Rho; rod) among recoverin-positive cells in the pars plana (H) are presented. Note that the abrupt alteration in the cone-rod ratio signified the cilioretinal border. E, I: Rare cells positive for both recoverin and BrdU were identified in the pars plana of MNU-treated mice (E; n=8); such cells were never observed in control mice (I; n=12). G: At P37, increased numbers of recoverin-positive cells were observed in the pars plana of MNU-treated mice (n=8) as compared to the controls (n=12). For H, 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 rhodopsin and PNA were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the proportion of recoverin-positive cells that were also positive for rhodopsin (Rho) or PNA (PNA) or neither (Rcv) per animal. Average proportion (%; mean±SEM) of cells for each of the three categories were determined from 8 animals. For F, G, and I, the number of cells positive for BrdU, recoverin, or both within 320 µm width of the pars plana were determined from an optical scan (10.0 μm thick). Values from three independent images randomly obtained from a same eye were averaged. Data extracted from totals of 8 MNU-treated mice and 12 control mice were statistically processed and were presented as means±SEM A and B are thick scans merged from 2 scans (each scan was 10.0 μm thick) while C is presented as a thin scan (a single scan 3.9 μm thick). D and E are also thick scans but merged from 3 scans (each scan was 10.0 μm thick). Scale bar equals 25 μm in C-E and 50 μm in A and B. Abbreviation: recoverin (Rcv).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2629738&req=5

f8: Neurogenesis in the pars plana in mice with MNU-induced photoreceptor degeneration. Double-headed arrows indicate the pars plana. MNU was injected at P30 and BrdU (50 mg/kg) was injected at P32. A, B, F: At P37, increased numbers of BrdU-positive cells were observed in the pars plana of MNU-treated mice (A) compared to untreated controls (B), which is summarized in F. Note that BrdU-positive cells in a linear alignment in the retina of B were blood cells in retinal vessels. C: Representative image of a cell positive for both recoverin and BrdU (arrowhead) in the peripheral retina at P40. D,H: Proportions of cells positive for PNA (cone) or rhodopsin (Rho; rod) among recoverin-positive cells in the pars plana (H) are presented. Note that the abrupt alteration in the cone-rod ratio signified the cilioretinal border. E, I: Rare cells positive for both recoverin and BrdU were identified in the pars plana of MNU-treated mice (E; n=8); such cells were never observed in control mice (I; n=12). G: At P37, increased numbers of recoverin-positive cells were observed in the pars plana of MNU-treated mice (n=8) as compared to the controls (n=12). For H, 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 rhodopsin and PNA were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the proportion of recoverin-positive cells that were also positive for rhodopsin (Rho) or PNA (PNA) or neither (Rcv) per animal. Average proportion (%; mean±SEM) of cells for each of the three categories were determined from 8 animals. For F, G, and I, the number of cells positive for BrdU, recoverin, or both within 320 µm width of the pars plana were determined from an optical scan (10.0 μm thick). Values from three independent images randomly obtained from a same eye were averaged. Data extracted from totals of 8 MNU-treated mice and 12 control mice were statistically processed and were presented as means±SEM A and B are thick scans merged from 2 scans (each scan was 10.0 μm thick) while C is presented as a thin scan (a single scan 3.9 μm thick). D and E are also thick scans but merged from 3 scans (each scan was 10.0 μm thick). Scale bar equals 25 μm in C-E and 50 μm in A and B. 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