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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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Presence of proliferating cells in the pars plana that escaped BrdU labeling and detection. The eyes were enucleated 3 or 11 h after intraperitoneal injection of BrdU (50 mg/kg) into P12 rd1 mice. Cilioretinal flat-mounts were stained with antibodies against BrdU and Ki-67, both of which are markers for cells proliferation. Double-headed arrows indicate the pars plana. A: At 3 h after BrdU injection, the cells positive for BrdU incorporation grossly matched those immunopositive for Ki-67 in the pars plana. B: By 11 h after BrdU injection, clusters of cells positive for Ki-67, but negative for BrdU (arrowheads), aligned circumferentially in selected areas of the pars plana. No such cluster was seen in the pars plicata or retina. A and B are presented as a thin scan (a single scan 10.0 μm thick). C: The proportion (%) of cells negative for BrdU but positive for Ki-67 (BrdU-Ki-67+) relative to BrdU-positive cells (BrdU+) are presented to evaluate the presence of mitotic cells that escaped BrdU labeling or detection. An increased percentage of BrdU-Ki-67+ cells was found in the pars plana 11 h after BrdU injection compared to 3 h after (4.60-fold; means±SEM). After the number of BrdU-positive cells within 320 µm width of the pars plana were determined from a single optical scan (10.0 μm thick), the number of those immunopositive for Ki-67, but negative for BrdU, were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the relative proportion of Ki-67-positive and BrdU-negative cells versus BrdU-positive cells per animal. Average proportion (%; mean±SEM) were determined from 8 animals each for P12 rd1 mice sacrificed 3 and 11 h after BrdU injection. Scale bar equals 50 μm.
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f7: Presence of proliferating cells in the pars plana that escaped BrdU labeling and detection. The eyes were enucleated 3 or 11 h after intraperitoneal injection of BrdU (50 mg/kg) into P12 rd1 mice. Cilioretinal flat-mounts were stained with antibodies against BrdU and Ki-67, both of which are markers for cells proliferation. Double-headed arrows indicate the pars plana. A: At 3 h after BrdU injection, the cells positive for BrdU incorporation grossly matched those immunopositive for Ki-67 in the pars plana. B: By 11 h after BrdU injection, clusters of cells positive for Ki-67, but negative for BrdU (arrowheads), aligned circumferentially in selected areas of the pars plana. No such cluster was seen in the pars plicata or retina. A and B are presented as a thin scan (a single scan 10.0 μm thick). C: The proportion (%) of cells negative for BrdU but positive for Ki-67 (BrdU-Ki-67+) relative to BrdU-positive cells (BrdU+) are presented to evaluate the presence of mitotic cells that escaped BrdU labeling or detection. An increased percentage of BrdU-Ki-67+ cells was found in the pars plana 11 h after BrdU injection compared to 3 h after (4.60-fold; means±SEM). After the number of BrdU-positive cells within 320 µm width of the pars plana were determined from a single optical scan (10.0 μm thick), the number of those immunopositive for Ki-67, but negative for BrdU, were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the relative proportion of Ki-67-positive and BrdU-negative cells versus BrdU-positive cells per animal. Average proportion (%; mean±SEM) were determined from 8 animals each for P12 rd1 mice sacrificed 3 and 11 h after BrdU injection. Scale bar equals 50 μm.

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)

Presence of proliferating cells in the pars plana that escaped BrdU labeling and detection. The eyes were enucleated 3 or 11 h after intraperitoneal injection of BrdU (50 mg/kg) into P12 rd1 mice. Cilioretinal flat-mounts were stained with antibodies against BrdU and Ki-67, both of which are markers for cells proliferation. Double-headed arrows indicate the pars plana. A: At 3 h after BrdU injection, the cells positive for BrdU incorporation grossly matched those immunopositive for Ki-67 in the pars plana. B: By 11 h after BrdU injection, clusters of cells positive for Ki-67, but negative for BrdU (arrowheads), aligned circumferentially in selected areas of the pars plana. No such cluster was seen in the pars plicata or retina. A and B are presented as a thin scan (a single scan 10.0 μm thick). C: The proportion (%) of cells negative for BrdU but positive for Ki-67 (BrdU-Ki-67+) relative to BrdU-positive cells (BrdU+) are presented to evaluate the presence of mitotic cells that escaped BrdU labeling or detection. An increased percentage of BrdU-Ki-67+ cells was found in the pars plana 11 h after BrdU injection compared to 3 h after (4.60-fold; means±SEM). After the number of BrdU-positive cells within 320 µm width of the pars plana were determined from a single optical scan (10.0 μm thick), the number of those immunopositive for Ki-67, but negative for BrdU, were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the relative proportion of Ki-67-positive and BrdU-negative cells versus BrdU-positive cells per animal. Average proportion (%; mean±SEM) were determined from 8 animals each for P12 rd1 mice sacrificed 3 and 11 h after BrdU injection. Scale bar equals 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Presence of proliferating cells in the pars plana that escaped BrdU labeling and detection. The eyes were enucleated 3 or 11 h after intraperitoneal injection of BrdU (50 mg/kg) into P12 rd1 mice. Cilioretinal flat-mounts were stained with antibodies against BrdU and Ki-67, both of which are markers for cells proliferation. Double-headed arrows indicate the pars plana. A: At 3 h after BrdU injection, the cells positive for BrdU incorporation grossly matched those immunopositive for Ki-67 in the pars plana. B: By 11 h after BrdU injection, clusters of cells positive for Ki-67, but negative for BrdU (arrowheads), aligned circumferentially in selected areas of the pars plana. No such cluster was seen in the pars plicata or retina. A and B are presented as a thin scan (a single scan 10.0 μm thick). C: The proportion (%) of cells negative for BrdU but positive for Ki-67 (BrdU-Ki-67+) relative to BrdU-positive cells (BrdU+) are presented to evaluate the presence of mitotic cells that escaped BrdU labeling or detection. An increased percentage of BrdU-Ki-67+ cells was found in the pars plana 11 h after BrdU injection compared to 3 h after (4.60-fold; means±SEM). After the number of BrdU-positive cells within 320 µm width of the pars plana were determined from a single optical scan (10.0 μm thick), the number of those immunopositive for Ki-67, but negative for BrdU, were determined from the same image. Three independent images randomly obtained from the same eye were analyzed to calculate the relative proportion of Ki-67-positive and BrdU-negative cells versus BrdU-positive cells per animal. Average proportion (%; mean±SEM) were determined from 8 animals each for P12 rd1 mice sacrificed 3 and 11 h after BrdU injection. Scale bar equals 50 μm.
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