Anat Cell Biol.  2010 Sep;43(3):218-229. 10.5115/acb.2010.43.3.218.

Changes in transcript and protein levels of calbindin D28k, calretinin and parvalbumin, and numbers of neuronal populations expressing these proteins in an ischemia model of rat retina

• Kim SA
• Jeon JH
• Son MJ
• Cha J
• Chun MH
• Kim IB

• KMID: 2168877
• DOI: http://doi.org/10.5115/acb.2010.43.3.218

Abstract

Excessive calcium is thought to be a critical step in various neurodegenerative processes including ischemia. Calbindin D28k (CB), calretinin (CR), and parvalbumin (PV), members of the EF-hand calcium-binding protein family, are thought to play a neuroprotective role in various pathologic conditions by serving as a buffer against excessive calcium. The expression of CB, PV and CR in the ischemic rat retina induced by increasing intraocular pressure was investigated at the transcript and protein levels, by means of the quantitative real-time reverse transcription-polymerase chain reaction, western blot and immunohistochemistry. The transcript and protein levels of CB, which is strongly expressed in the horizontal cells in both normal and affected retinas, were not changed significantly and the number of CB-expressing horizontal cells remained unchanged throughout the experimental period 8 weeks after ischemia/reperfusion injury. At both the transcript and protein levels, however, CR, which is strongly expressed in several types of amacrine, ganglion, and displaced amacrine cells in both normal and affected retinas, was decreased. CR-expressing ganglion cell number was particularly decreased in ischemic retinas. Similar to the CR, PV transcript and protein levels, and PV-expressing AII amacrine cell number were decreased. Interestingly, in ischemic retinas PV was transiently expressed in putative cone bipolar cell types possibly those that connect with AII amacrine cells via gap junctions. These results suggest that these three calcium binding proteins may play different neuroprotective roles in ischemic insult by their ability to buffer calcium in the rat retina.

Keyword

Calbindin; calretinin; ischemia; parvalbumin; rat retina

MeSH Terms

Amacrine Cells
Animals
Blotting, Western
Calcium
Calcium-Binding Protein, Vitamin D-Dependent
Calcium-Binding Proteins
Cell Count
Ganglion Cysts
Gap Junctions
Humans
Immunohistochemistry
Intraocular Pressure
Ischemia
Neurons
Proteins
Rats
Retina
Calcium
Calcium-Binding Protein, Vitamin D-Dependent
Calcium-Binding Proteins
Proteins

Figure

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  Fig. 1 Expression levels of calbindin (CB), calretinin (CR), and parvalbumin (PV) transcripts after ischemia/reperfusion injury in the rat retina. Error bars indicate standard deviation. *P<0.05; Student's t test.

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  Fig. 2 Expression levels of CB, CR, and PV proteins after ischemia/reperfusion injury in the rat retina. In the upper panel, Western blottings with CB (A), CR (B), and PV (C) antibodies demonstrate a single band at a molecular weight of 28-kDa, 29-kDa, and 12-kDa, respectively. Lower panel shows the results of densitometric analyses of Western blots shown in the upper panel.

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  Fig. 3 Changes in the number of CB-expressing neurons after ischemia/reperfusion injury in the rat retina. A~C: confocal micrographs taken from vertical vibratome sections (40-µm-thick) of the normal rat retina (A) and retinas at 4 wks (B), and 8 wks (C) after ischemia/reperfusion injury, processed for CB immunoreactivities. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 50 µm. D, E: confocal micrographs taken from wholemounts of the normal rat retina (D) and retina 8 wks (E) after ischemia/reperfusion, processed for CB immunoreactivities. The figures (5 µm-thick) were formed by stacking optical sections focusing on the OPL to the outer part of the INL. Scale bar, 50 µm. F: quantitative assessment of the number of CB-expressing horizontal cells per mm2 in normal retina (CTR) and retinas at 1 wk, 4 wks and 8 wks after ischemia/reperfusion injury. Error bars indicate standard deviation. *P<0.05; Student's t test.

• 

  Fig. 4 Changes in the number of CR-expressing neurons after ischemia/reperfusion injury in the rat retina. A~C: confocal micrographs taken from vertical vibratome sections (40-µm-thick) of the normal rat retina (A) and retinas at 4 wks (B), and 8 wks (C) after ischemia/reperfusion injury, processed for CR immunoreactivities. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 50 µm. D, E: confocal micrographs taken from wholemounts of the normal rat retina (D) and retina 8 wks (E) after ischemia/reperfusion, processed for CR immunoreactivities. The figures (8 µm-thick) were formed by stacking optical sections throughout the GCL. Scale bar, 50 µm. F: quantitative assessment of the number of CR-expressing ganglion cells per mm2 in normal retina (CTR) and retinas at 1 wk, 4 wks and 8 wks after ischemia/reperfusion injury. Error bars indicate standard deviation. *P<0.05; Student's t test.

• 

  Fig. 5 Changes in the number of PV-expressing neurons after ischemia/reperfusion injury in the rat retina. A~F: confocal micrographs taken from vertical vibratome sections (40-µm-thick) of the normal rat retina (A) and retinas at 3 days (B), 1 wk (C), 2 wks (D), 4 wks (E), and 8 wks (F) after ischemia/reperfusion injury, processed for PV immunoreactivities. Arrows indicate a new PV-expressing population. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar, 50 µm. G, H: confocal micrographs taken from wholemounts of the normal rat retina (G) and retina 8 wks (H) after ischemia/reperfusion, processed for PV immunoreactivities. The figures (8 µm-thick) were formed by stacking optical sections throughout the GCL. Scale bar, 50 µm. I: quantitative assessment of the number of PV-expressing AII amacrine cells per mm2 in normal retina (CTR) and retinas at 1 wk, 4 wks and 8 wks after ischemia/reperfusion injury. Error bars indicate standard deviation. *P<0.05; Student's t test.

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