Korean J Ophthalmol.  2012 Jun;26(3):203-211. 10.3341/kjo.2012.26.3.203.

Expression of the Na(+)-K(+)-2Cl(-)-Cotransporter 2 in the Normal and Pressure-Induced Ischemic Rat Retina

• Kim DH
• Ahn MD

• KMID: 1376127
• DOI: http://doi.org/10.3341/kjo.2012.26.3.203

Abstract

PURPOSE
To evaluate the expression of the Na(+)-K(+)-2Cl(-)-cotransporter 2 (NKCC2) in the ischemic rat retina.
METHODS
Retinal ischemia was induced by pressures 90 to 120 mmHg, above systemic systolic pressure. Immunohistochemistry and western blot analysis were performed.
RESULTS
NKCC2 is expressed in the normal retina and its expression is increased by ischemia caused by intraocular pressure elevation. NKCC2 immunoreactivity was observed mainly in axon bundles of ganglion cells and horizontal cell processes in the retina. NKCC2 expression continuously increased with a peak value 3 days (to 415% of normal levels) after ischemic injury, and then gradually decreased to 314% of controls until 2 weeks post injury. The mean density of NKCC2-labeled ganglion cells per mm2 changed from 1,255 +/- 109 in normal retinas to 391 +/- 49 and 185 +/- 37 at 3 days and 2 weeks after ischemia, respectively (p < 0.05), implying cell death of ganglion cells labeled with NKCC2.
CONCLUSIONS
Taken together, these results suggest that NKCC2, which is expressed in retinal ganglion and horizontal cells, may contribute to cell death by ischemic injury in the retina, although the molecular mechanisms involved remain to be clarified.

Keyword

Ischemia; Na(+)-K(+)-2Cl(-)-cotransporter 2; Retinal ganglion cells

MeSH Terms

Animals
Blotting, Western
Disease Models, Animal
Immunohistochemistry
Intraocular Pressure
Ischemia/etiology/*metabolism
Male
Microscopy, Confocal
Ocular Hypertension/*complications/metabolism/physiopathology
Rats
Rats, Sprague-Dawley
Retinal Diseases/etiology/*metabolism
Retinal Ganglion Cells/*metabolism/pathology
Sodium-Potassium-Chloride Symporters/*biosynthesis

Figure

• 

  Fig. 1 Immunoblot analysis of the Na+-K+-2Cl--cotransporter 2 (NKCC2) protein levels in normal and ischemic rat retina. (A) Immunoblot stained for the NKCC2 demonstrating a single band at 120 to 130 kDa for all time points tested: normal retina (lane 1), 1 day (lane 2), 3 days (lane 3), 7 days (lane 4), and 14 days (lane 5) after ischemia/reperfusion. (B) Densitometric analysis of immunoblots as shown in (A). The NKCC2 level was upregulated to approximately 115% of control by 1 day and to approximately 415% by 3 days. The NKCC2 levels decreased to 275% and 313% at 7 days and 14 days of ischemia/reperfusion, respectively. Asterisk represents a significant NKCC2 expression increase (p < 0.05, one way ANOVA).

• 

  Fig. 2 Immunocytochemical staining for the Na+-K+-2Cl--cotransporter 2 (NKCC2) in normal and ischemic rat retina. Photomicrographs show 50-µm-thick sections processed for immunocytochemistry. (A) Normal retina. (B,C,D,E) Ischemic retina 1, 3, 7, and 14 days after ischemia/reperfusion, respectively. Labeling for the NKCC2 is present in both the ganglion and outer plexiform layers. After ischemia/reperfusion, retinal ganglion cells decrease and marked axonal sprouting in the outer plexiform layer was observed. In the ganglion cell layer, the ganglion cell axon bundles (asterisk) and ganglion cell body (arrow) are seen. Arrow head indicates the horizontal cell processes in the outer plexiform layer. ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer. Scale bar = 50 µm.

• 

  Fig. 3 Confocal micrographs taken from a vertical section of normal retina (A,B,C) and ischemic retina 2 weeks after injury (D,E,F). Tissues were processed for Na+-K+-2Cl--cotransporter 2 (NKCC2) (A,D) and Thy1.1 (B,E) immunohistochemistry. The NKCC2 labeled ganglion cells are immunofluorescent using Cy3-conjugated secondary antibody, and Thy1.1 was visualized using a FITC-conjugated secondary antibody. The NKCC2 demonstrates good immunoreactivity, comparable to Thy1.1 (C,F) Merged picture shows that Thy1.1 immunoreactive profiles show NKCC2 immunoreactivity (yellow). ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer. Scale bar = 50 µm.

• 

  Fig. 4 Confocal micrographs taken from a vertical section of normal retina (A,B,C) and ischemic retina 2 weeks after injury (D,E,F). Tissues were processed for Na+-K+-2Cl--cotransporter 2 (NKCC2) (A,D) and calbindin (B,E) immunohistochemistry. The NKCC2 labeled ganglion cells are immunofluorescent using Cy3-conjugated secondary antibody and calbindin was visualized using a FITC-conjugated secondary antibody. (C,F) Merged picture shows that calbindin immunoreactive profiles show NKCC2 immunoreactivity (yellow). After ischemia/reperfusion, the NKCC2 immunoreactivity increased and the number of horizontal cells did not decrease. ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer. Scale bar = 50 µm.

• 

  Fig. 5 Light micrographs taken from whole-mount retinas processed for the Na+-K+-2Cl--cotransporter 2 (NKCC2) in normal retina (A) and 3 (B) and 7 days (C) after ischemia/reperfusion. The focus is at the ganglion cell layer. In normal retina, the NKCC2 labeled ganglion cells (arrows) and axon bundles are clearly distinguishable. In the ischemic retina, the number of the NKCC2 labeled ganglion cells decreased compared with the normal retina. Scale bar = 50 µm.

• 

  Fig. 6 Densities of the Na+-K+-2Cl--cotransporter 2 (NKCC2) immunoresponsive retinal ganglion cells after transient ischemia/reperfusion (cells/mm2). Densities decreased significantly 3 days and 2 weeks after injury. Asterisk represents a significant decrease of the NKCC2-labeled ganglion cell densities (p < 0.05, one way ANOVA).

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