Anat Cell Biol.  2011 Mar;44(1):25-34. 10.5115/acb.2011.44.1.25.

Immunochemical changes of calbindin, calretinin and SMI32 in ischemic retinas induced by increase of intraocular pressure and by middle cerebral artery occlusion

• Lee JH
• Shin JM
• Shin YJ
• Chun MH
• Oh SJ

• KMID: 2168865
• DOI: http://doi.org/10.5115/acb.2011.44.1.25

Abstract

The reaction of neuroactive substances to ischemic conditions in the rat retina evoked by different methods was immunochemically evaluated in adult Sprague-Dawley rats. Ocular ischemic conditions were unilaterally produced by elevating intraocular pressure (EIOP) or by middle cerebral artery occlusion (MCAO). Two EF-hand calcium binding proteins, calbindin D28K (CB) and calretinin (CR), in the normal retina showed similar immunolocalization, such as the amacrine and displaced amacrine cells, the ganglion cells, and their processes, particularly CB in horizontal cells. CB immunoreactive neurons in the ganglion cell layer in both types of ischemic retinas were more reduced in number than CR neurons compared to those in a normal retina. The CB protein level in both ischemic retinas was reduced to 60-80% of normal. The CR protein level in MCAO retinas was reduced to about 80% of normal but increased gradually to the normal value, whereas that in the EIOP showed a gradual reduction and a slight recovery. SMI32 immunoreactivity, which detects a dephosphorylated epitope of neurofilaments-M and -H, appeared in the axon bundles of ganglion cells in the innermost nerve fiber layer of normal retinas. The reactivity in the nerve fiber bundles appeared to only increase slightly in EIOP retinas, whereas a moderate increase occurred in MCAO retinas. The SMI32 protein level in MCAO retinas showed a gradual increasing tendency, whereas that in the EIOP showed a slight fluctuation. Interestingly, the MCAO retinas showed additional SMI32 immunoreactivity in the cell soma of presumed ganglion cells, whereas that of EIOP appeared in the Muller proximal radial fibers. Glial fibrillary acidic protein (GFAP) immunoreactivity appeared in the astrocytes located in the nerve fiber layer of normal retinas. Additional GFAP immunoreactivity appeared in the Muller glial fibers deep in EIOP retinas and at the proximal end in MCAO retinas. These findings suggest that the neurons in the ganglion cell layer undergo degenerative changes in response to ischemia, although EIOP retinas represented a remarkable Muller glial reaction, whereas MCAO retinas had only a small-scaled axonal transport disturbance.

Keyword

Elevation of intraocular pressure; Middle cerebral artery occlusion; Calcium-binding protein; SMI32; Retina

MeSH Terms

Adult
Amacrine Cells
Animals
Astrocytes
Axonal Transport
Axons
Calcium-Binding Protein, Vitamin D-Dependent
Calcium-Binding Proteins
Carisoprodol
Ganglion Cysts
Glial Fibrillary Acidic Protein
Humans
Infarction, Middle Cerebral Artery
Intraocular Pressure
Ischemia
Middle Cerebral Artery
Nerve Fibers
Neurons
Rats
Rats, Sprague-Dawley
Reference Values
Retina
Calcium-Binding Protein, Vitamin D-Dependent
Calcium-Binding Proteins
Carisoprodol
Glial Fibrillary Acidic Protein

Figure

• 

  Fig. 1 Light micrographs showing immunohistochemical changes in the retina taken from normal (A), 3 days (B) and 14 days (C) after elevation of intraocular pressure, and 1 day (D), 3 days (E), and 7 days (F) after middle cerebral artery occlusion, which were processed for anti-calbindin immunohistochemistry. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar=50 µm.

• 

  Fig. 2 Quantitative analysis of the number of calbindin D28K-immunoreactive neurons in the ganglion cell layer in the retinas at normal (N) and at 1 day (1D), 3 days (3D), 7 days (7D), and 14 days (14D) after both elevation of intraocular pressure (EIOP) and middle cerebral artery occlusion (MCAO), which is represented as mean cell number and standard deviation appearing per 1 mm length of 5 µm-thick vertical retinal section. *P<0.05; Student's t-test.

• 

  Fig. 3 Light micrographs showing immunohistochemical changes in the retina taken from normal (A), 1 day (B) and 14 days (C) after elevation of intraocular pressure, and 3 days (D), 7 days (E), and 14 days (F) after middle cerebral artery occlusion, which were processed for anti-calretinin immunohistochemistry. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar=50 µm.

• 

  Fig. 4 Quantitative analysis of the number of calretinin-immunoreactive neurons in the ganglion cell layer in the retinas at normal (N) and at 1 day (1D), 3 days (3D), 7 days (7D), and 14 days (14D) after both elevation of intraocular pressure (EIOP) and middle cerebral artery occlusion (MCAO). Data represented as mean cell number and standard deviation appearing per 1 mm length of 5 µm-thick vertical retinal section. *P<0.05; Student's t-test.

• 

  Fig. 5 Light micrographs showing immunohistochemical changes in the retina taken from 3 days (A) and 7 days (B) after elevation of intraocular pressure, and 3 days (C) and 7 days (D) after middle cerebral artery occlusion, which were processed for anti-SMI32 immunohistochemistry. Arrows indicate SMI-immunoreactive ganglion cells in the ganglion cell layer. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bar=50 µm.

• 

  Fig. 6 Densitometric analyses of immunoblots of calbindin D28K (A), calretinin (B), and SMI32 (C) protein levels in the retinas at normal (N) and at 1 day (1D), 3 days (3D), 7 days (7D), and 14 days (14D) after both elevation of intraocular pressure (EIOP) and middle cerebral artery occlusion (MCAO). Data are shown as means±standard deviation. *P<0.05, Student's t-test.

• 

  Fig. 7 Light micrographs showing immunohistochemical changes in the retina taken from normal (A), 7 days (B) after elevation of intraocular pressure, and 3 days (C) and 7 days (D) after middle cerebral artery occlusion, which were processed for anti-glial fibrillary acidic protein immunohistochemistry. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar=50 µm.

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