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Mature retinal pigment epithelium cells are retained in the cell cycle and proliferate in vivo.

Al-Hussaini H, Kam JH, Vugler A, Semo M, Jeffery G - Mol. Vis. (2008)

Bottom Line: These cells were negative for Caspase 3, hence were not apoptotic.Ki67-positive cells were also seen in human RPE.Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ophthalmology, University College London, London, United Kingdom.

ABSTRACT

Purpose: To investigate the capacity of mature retinal pigment epithelium (RPE) cells to enter the cell cycle in vivo using a range of RPE-specific and proliferative specific markers in both pigmented and albino rats.

Methods: Whole-mounted retinas of both Dark Agouti and albino rats were immunolabeled with cell cycle markers Ki67 or PCNA and double labeled with RPE cell marker RPE65 or CRALBP. The number and distribution of these cells was mapped. An additional group of Dark Agouti rats were given repeated intraperitoneal injections of Bromodeoxyuridine (BrdU )for 20 days and then sacrificed 30 days later. The retinas were then processed for BrdU detection and Otx, a RPE cell-specific marker. For comparison, human RPE tissue from a postmortem donor was also labeled for Ki67.

Results: In both pigmentation phenotypes, a subpopulation of mature RPE cells in the periphery were positive for both cell cycle markers. These cells were negative for Caspase 3, hence were not apoptotic. Ki67-positive cells were also seen in human RPE. Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate. There was a ten fold increase in the number of RPE cells positive for cell cycle markers in albino (approximately 200 cells) compared to pigmented rats (approximately 20 cells).

Conclusions: Peripheral RPE cells in rats have the capacity to enter the cell cycle and complete cellular division.

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Related in: MedlinePlus

BrdU labeling in mature RPE cells. A and B: Shows a binucleated labeled cell with BrdU on red channel and Otx green channel. C shows two adjacent mono-nucleated cells that are labeled with BrdU. Scale bar equals 20 µm. D shows an outline drawing on which the location of RPE cells labeled with Ki67 and BrdU are marked. The diagram shows the distribution of positive BrdU-labeled binucleated (black dots) and mononucleated (black circle) cells. Only a small number of the BrdU-labeled cells were more centrally located than those labeled for Ki67. The mononucleated cells were almost always found in pairs of close proximity. The red dots represent the number and distribution of Ki67 positive RPE cells. While these largely overlap with the BrdU labeled population of RPE cells, they tend to occupy a slightly more peripheral location. The scale bar represents 2.5 mm.
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f6: BrdU labeling in mature RPE cells. A and B: Shows a binucleated labeled cell with BrdU on red channel and Otx green channel. C shows two adjacent mono-nucleated cells that are labeled with BrdU. Scale bar equals 20 µm. D shows an outline drawing on which the location of RPE cells labeled with Ki67 and BrdU are marked. The diagram shows the distribution of positive BrdU-labeled binucleated (black dots) and mononucleated (black circle) cells. Only a small number of the BrdU-labeled cells were more centrally located than those labeled for Ki67. The mononucleated cells were almost always found in pairs of close proximity. The red dots represent the number and distribution of Ki67 positive RPE cells. While these largely overlap with the BrdU labeled population of RPE cells, they tend to occupy a slightly more peripheral location. The scale bar represents 2.5 mm.

Mentions: Are Ki67-positive cells in the periphery dividing or undergoing only nuclear division to become binucleated? Three lines of evidence support the notion that at least some of these cells are going through full cell division. First, if the nuclei of RPE cells are dividing but not the cell, then the number of binucleated cells should increase with age in the periphery. When the number of RPE binucleated cells at P20 and P60 were compared in the periphery, no significant difference was found in their number between the two ages (p>0.5). Second, there were cells identified that were Ki67 positive that possessed two labeled nuclei and appeared to be passing through full cell division, establishing a cytoplasmic membrane between their nuclei. These cells also appeared to be irregular within the geometric configuration of the regular RPE (Figure 5). Ki67 and PCNA are only associated with proliferation as they are simply cell cycle. However, the critical factor in favor of this resulting in cell division is that BrdU was detected in peripheral retinal cells. These were a mixed population with some having a single nucleus and others being binucleated. Cells with a single nucleus were commonly in adjacent pairs (Figure 6). Multiple injections of BrdU given at 3 and 12 h intervals failed to significantly increase the size of this population compared with animals given a single pulse. However, those given at 24 h intervals over 5 days did significantly expand the number of positive cells compared with all other groups (ANOVA=0.0001, Dunnett's Multiple Comparison Test=0.001, Figure 7). Hence, it is likely that the cell cycle rate is very slow.


Mature retinal pigment epithelium cells are retained in the cell cycle and proliferate in vivo.

Al-Hussaini H, Kam JH, Vugler A, Semo M, Jeffery G - Mol. Vis. (2008)

BrdU labeling in mature RPE cells. A and B: Shows a binucleated labeled cell with BrdU on red channel and Otx green channel. C shows two adjacent mono-nucleated cells that are labeled with BrdU. Scale bar equals 20 µm. D shows an outline drawing on which the location of RPE cells labeled with Ki67 and BrdU are marked. The diagram shows the distribution of positive BrdU-labeled binucleated (black dots) and mononucleated (black circle) cells. Only a small number of the BrdU-labeled cells were more centrally located than those labeled for Ki67. The mononucleated cells were almost always found in pairs of close proximity. The red dots represent the number and distribution of Ki67 positive RPE cells. While these largely overlap with the BrdU labeled population of RPE cells, they tend to occupy a slightly more peripheral location. The scale bar represents 2.5 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562424&req=5

f6: BrdU labeling in mature RPE cells. A and B: Shows a binucleated labeled cell with BrdU on red channel and Otx green channel. C shows two adjacent mono-nucleated cells that are labeled with BrdU. Scale bar equals 20 µm. D shows an outline drawing on which the location of RPE cells labeled with Ki67 and BrdU are marked. The diagram shows the distribution of positive BrdU-labeled binucleated (black dots) and mononucleated (black circle) cells. Only a small number of the BrdU-labeled cells were more centrally located than those labeled for Ki67. The mononucleated cells were almost always found in pairs of close proximity. The red dots represent the number and distribution of Ki67 positive RPE cells. While these largely overlap with the BrdU labeled population of RPE cells, they tend to occupy a slightly more peripheral location. The scale bar represents 2.5 mm.
Mentions: Are Ki67-positive cells in the periphery dividing or undergoing only nuclear division to become binucleated? Three lines of evidence support the notion that at least some of these cells are going through full cell division. First, if the nuclei of RPE cells are dividing but not the cell, then the number of binucleated cells should increase with age in the periphery. When the number of RPE binucleated cells at P20 and P60 were compared in the periphery, no significant difference was found in their number between the two ages (p>0.5). Second, there were cells identified that were Ki67 positive that possessed two labeled nuclei and appeared to be passing through full cell division, establishing a cytoplasmic membrane between their nuclei. These cells also appeared to be irregular within the geometric configuration of the regular RPE (Figure 5). Ki67 and PCNA are only associated with proliferation as they are simply cell cycle. However, the critical factor in favor of this resulting in cell division is that BrdU was detected in peripheral retinal cells. These were a mixed population with some having a single nucleus and others being binucleated. Cells with a single nucleus were commonly in adjacent pairs (Figure 6). Multiple injections of BrdU given at 3 and 12 h intervals failed to significantly increase the size of this population compared with animals given a single pulse. However, those given at 24 h intervals over 5 days did significantly expand the number of positive cells compared with all other groups (ANOVA=0.0001, Dunnett's Multiple Comparison Test=0.001, Figure 7). Hence, it is likely that the cell cycle rate is very slow.

Bottom Line: These cells were negative for Caspase 3, hence were not apoptotic.Ki67-positive cells were also seen in human RPE.Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ophthalmology, University College London, London, United Kingdom.

ABSTRACT

Purpose: To investigate the capacity of mature retinal pigment epithelium (RPE) cells to enter the cell cycle in vivo using a range of RPE-specific and proliferative specific markers in both pigmented and albino rats.

Methods: Whole-mounted retinas of both Dark Agouti and albino rats were immunolabeled with cell cycle markers Ki67 or PCNA and double labeled with RPE cell marker RPE65 or CRALBP. The number and distribution of these cells was mapped. An additional group of Dark Agouti rats were given repeated intraperitoneal injections of Bromodeoxyuridine (BrdU )for 20 days and then sacrificed 30 days later. The retinas were then processed for BrdU detection and Otx, a RPE cell-specific marker. For comparison, human RPE tissue from a postmortem donor was also labeled for Ki67.

Results: In both pigmentation phenotypes, a subpopulation of mature RPE cells in the periphery were positive for both cell cycle markers. These cells were negative for Caspase 3, hence were not apoptotic. Ki67-positive cells were also seen in human RPE. Further, many cells positive for BrdU were identified in similar retinal regions, confirming that RPE cells not only enter the cell cycle but also divide, albeit at a slow cell cycle rate. There was a ten fold increase in the number of RPE cells positive for cell cycle markers in albino (approximately 200 cells) compared to pigmented rats (approximately 20 cells).

Conclusions: Peripheral RPE cells in rats have the capacity to enter the cell cycle and complete cellular division.

Show MeSH
Related in: MedlinePlus