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Microglia in mouse retina contralateral to experimental glaucoma exhibit multiple signs of activation in all retinal layers.

Rojas B, Gallego BI, Ramírez AI, Salazar JJ, de Hoz R, Valiente-Soriano FJ, Avilés-Trigueros M, Villegas-Perez MP, Vidal-Sanz M, Triviño A, Ramírez JM - J Neuroinflammation (2014)

Bottom Line: In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL.Such activation extended beyond the GCL, involving all retinal layers.Differences between the two eyes could help to elucidate glaucoma pathophysiology.

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Affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Facultad de Medicina, Pab VI, 4a, Avenida Complutense s/n, Universidad Complutense de Madrid, 28040 Madrid, Spain. ramirezs@med.ucm.es.

ABSTRACT

Background: Glaucomatous optic neuropathy, a leading cause of blindness, can progress despite control of intraocular pressure - currently the main risk factor and target for treatment. Glaucoma progression shares mechanisms with neurodegenerative disease, including microglia activation. In the present model of ocular hypertension (OHT), we have recently described morphological signs of retinal microglia activation and MHC-II upregulation in both the untreated contralateral eyes and OHT eyes. By using immunostaining, we sought to analyze and quantify additional signs of microglia activation and differences depending on the retinal layer.

Methods: Two groups of adult Swiss mice were used: age-matched control (naïve, n = 12), and lasered (n = 12). In the lasered animals, both OHT eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1, MHC-II, CD68, CD86, and Ym1. The Iba-1+ cell number in the plexiform layers (PL) and the photoreceptor outer segment (OS), Iba-1+ arbor area in the PL, and area of the retina occupied by Iba-1+ cells in the nerve fiber layer-ganglion cell layer (NFL-GCL) were quantified.

Results: The main findings in contralateral eyes and OHT eyes were: i) ameboid microglia in the NFL-GCL and OS; ii) the retraction of processes in all retinal layers; iii) a higher level of branching in PL and in the OS; iv) soma displacement to the nearest cell layers in the PL and OS; v) the reorientation of processes in the OS; vi) MHC-II upregulation in all retinal layers; vii) increased CD68 immunostaining; and viii) CD86 immunolabeling in ameboid cells. In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL. In addition, rounded Iba-1+ CD86+ cells in the NFL-GCL, OS and Ym1+ cells, and rod-like microglia in the NFL-GCL were restricted to OHT eyes.

Conclusions: Several quantitative and qualitative signs of microglia activation are detected both in the contralateral and OHT eyes. Such activation extended beyond the GCL, involving all retinal layers. Differences between the two eyes could help to elucidate glaucoma pathophysiology.

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CD86 and Ym1 expression in the retina after 15 days of unilateral laser-induced OHT. Double immunostaining: Iba-1/CD86 (A-I) and MHC-II/Ym1 (J-L). Retinal whole-mount. In contralateral eyes (A-C) CD86 immunostaining was observed in some ameboid Iba-1+ cells in the OS (blank arrowhead) and NFL-GCL (inset). In OHT eyes (D-I) rounded Iba-1+ cells (arrow) and most ameboid Iba-1+ cells (blank arrowhead) were CD86+. Ym1 immunoreaction (J-L) was restricted to a few ameboid Iba-1+ cells (blank arrowhead) in the NFL-GCL of OHT eyes. (NFL-GCL: nerve fiber layer-ganglion cell layer; OHT: ocular hypertension; OS: photoreceptor outer segment).
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Figure 11: CD86 and Ym1 expression in the retina after 15 days of unilateral laser-induced OHT. Double immunostaining: Iba-1/CD86 (A-I) and MHC-II/Ym1 (J-L). Retinal whole-mount. In contralateral eyes (A-C) CD86 immunostaining was observed in some ameboid Iba-1+ cells in the OS (blank arrowhead) and NFL-GCL (inset). In OHT eyes (D-I) rounded Iba-1+ cells (arrow) and most ameboid Iba-1+ cells (blank arrowhead) were CD86+. Ym1 immunoreaction (J-L) was restricted to a few ameboid Iba-1+ cells (blank arrowhead) in the NFL-GCL of OHT eyes. (NFL-GCL: nerve fiber layer-ganglion cell layer; OHT: ocular hypertension; OS: photoreceptor outer segment).

Mentions: Both in contralateral (Figure 9B,E,H,K) and OHT eyes (Figure 9C,F,I,L), Iba-1+ cells showed upregulation in MHC-II expression in all retinal layers (Additional file 1). In contralateral eyes CD68 immunostaining was similar to naïve eyes except for a few ramified Iba-1+ cells in the IPL which had a patchy CD68+ immunostaining instead of the punctate immunostaining observed in naïve eyes (Additional file 1). In addition, ameboid Iba-1+ cells in the NFL-GCL (Figure 5A,B) and OS (Figure 10C,D) in contralateral eyes exhibited intense CD68 immunostaining. CD86 immunostaining was restricted to some ameboid Iba-1+ cells (Figure 11B,C; Additional file 1). No Ym1+ cells were observed (Additional file 1).


Microglia in mouse retina contralateral to experimental glaucoma exhibit multiple signs of activation in all retinal layers.

Rojas B, Gallego BI, Ramírez AI, Salazar JJ, de Hoz R, Valiente-Soriano FJ, Avilés-Trigueros M, Villegas-Perez MP, Vidal-Sanz M, Triviño A, Ramírez JM - J Neuroinflammation (2014)

CD86 and Ym1 expression in the retina after 15 days of unilateral laser-induced OHT. Double immunostaining: Iba-1/CD86 (A-I) and MHC-II/Ym1 (J-L). Retinal whole-mount. In contralateral eyes (A-C) CD86 immunostaining was observed in some ameboid Iba-1+ cells in the OS (blank arrowhead) and NFL-GCL (inset). In OHT eyes (D-I) rounded Iba-1+ cells (arrow) and most ameboid Iba-1+ cells (blank arrowhead) were CD86+. Ym1 immunoreaction (J-L) was restricted to a few ameboid Iba-1+ cells (blank arrowhead) in the NFL-GCL of OHT eyes. (NFL-GCL: nerve fiber layer-ganglion cell layer; OHT: ocular hypertension; OS: photoreceptor outer segment).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4128533&req=5

Figure 11: CD86 and Ym1 expression in the retina after 15 days of unilateral laser-induced OHT. Double immunostaining: Iba-1/CD86 (A-I) and MHC-II/Ym1 (J-L). Retinal whole-mount. In contralateral eyes (A-C) CD86 immunostaining was observed in some ameboid Iba-1+ cells in the OS (blank arrowhead) and NFL-GCL (inset). In OHT eyes (D-I) rounded Iba-1+ cells (arrow) and most ameboid Iba-1+ cells (blank arrowhead) were CD86+. Ym1 immunoreaction (J-L) was restricted to a few ameboid Iba-1+ cells (blank arrowhead) in the NFL-GCL of OHT eyes. (NFL-GCL: nerve fiber layer-ganglion cell layer; OHT: ocular hypertension; OS: photoreceptor outer segment).
Mentions: Both in contralateral (Figure 9B,E,H,K) and OHT eyes (Figure 9C,F,I,L), Iba-1+ cells showed upregulation in MHC-II expression in all retinal layers (Additional file 1). In contralateral eyes CD68 immunostaining was similar to naïve eyes except for a few ramified Iba-1+ cells in the IPL which had a patchy CD68+ immunostaining instead of the punctate immunostaining observed in naïve eyes (Additional file 1). In addition, ameboid Iba-1+ cells in the NFL-GCL (Figure 5A,B) and OS (Figure 10C,D) in contralateral eyes exhibited intense CD68 immunostaining. CD86 immunostaining was restricted to some ameboid Iba-1+ cells (Figure 11B,C; Additional file 1). No Ym1+ cells were observed (Additional file 1).

Bottom Line: In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL.Such activation extended beyond the GCL, involving all retinal layers.Differences between the two eyes could help to elucidate glaucoma pathophysiology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto de Investigaciones Oftalmológicas Ramón Castroviejo, Facultad de Medicina, Pab VI, 4a, Avenida Complutense s/n, Universidad Complutense de Madrid, 28040 Madrid, Spain. ramirezs@med.ucm.es.

ABSTRACT

Background: Glaucomatous optic neuropathy, a leading cause of blindness, can progress despite control of intraocular pressure - currently the main risk factor and target for treatment. Glaucoma progression shares mechanisms with neurodegenerative disease, including microglia activation. In the present model of ocular hypertension (OHT), we have recently described morphological signs of retinal microglia activation and MHC-II upregulation in both the untreated contralateral eyes and OHT eyes. By using immunostaining, we sought to analyze and quantify additional signs of microglia activation and differences depending on the retinal layer.

Methods: Two groups of adult Swiss mice were used: age-matched control (naïve, n = 12), and lasered (n = 12). In the lasered animals, both OHT eyes and contralateral eyes were analyzed. Retinal whole-mounts were immunostained with antibodies against Iba-1, MHC-II, CD68, CD86, and Ym1. The Iba-1+ cell number in the plexiform layers (PL) and the photoreceptor outer segment (OS), Iba-1+ arbor area in the PL, and area of the retina occupied by Iba-1+ cells in the nerve fiber layer-ganglion cell layer (NFL-GCL) were quantified.

Results: The main findings in contralateral eyes and OHT eyes were: i) ameboid microglia in the NFL-GCL and OS; ii) the retraction of processes in all retinal layers; iii) a higher level of branching in PL and in the OS; iv) soma displacement to the nearest cell layers in the PL and OS; v) the reorientation of processes in the OS; vi) MHC-II upregulation in all retinal layers; vii) increased CD68 immunostaining; and viii) CD86 immunolabeling in ameboid cells. In comparison with the control group, a significant increase in the microglial number in the PL, OS, and in the area occupied by Iba-1+ cells in the NFL-GCL, and significant reduction of the arbor area in the PL. In addition, rounded Iba-1+ CD86+ cells in the NFL-GCL, OS and Ym1+ cells, and rod-like microglia in the NFL-GCL were restricted to OHT eyes.

Conclusions: Several quantitative and qualitative signs of microglia activation are detected both in the contralateral and OHT eyes. Such activation extended beyond the GCL, involving all retinal layers. Differences between the two eyes could help to elucidate glaucoma pathophysiology.

Show MeSH
Related in: MedlinePlus