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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.

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.

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Related in: MedlinePlus

Retinal whole-mount. A: areas of retina selected for quantitative analysis of Iba-1+ cells. B: photomicrograph illustrating the method used for arbor-area quantification of Iba-1+ cells. A polygon was drawn manually by connecting the distal-most tips of the Iba-1+ cell processes.
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Figure 1: Retinal whole-mount. A: areas of retina selected for quantitative analysis of Iba-1+ cells. B: photomicrograph illustrating the method used for arbor-area quantification of Iba-1+ cells. A polygon was drawn manually by connecting the distal-most tips of the Iba-1+ cell processes.

Mentions: To determine the effect of OHT on Iba-1+ cells, we quantified these cells in the retinal whole-mounts of naïve (n = 6), contralateral (n = 9), and OHT eyes (n = 9). Twenty-four equivalent areas of the retina were consistently selected for each retinal whole-mount in both the vertical and horizontal meridians which cross the optic nerve (Figure 1A). Each complete meridian selected in the retinal whole-mount was analyzed using the motorized stage of the microscope to scan their whole extension along the X-Y axis, respectively. Thus, all subsequent fields analyzed were contiguous and were examined systematically to ensure that no portion of the retinal whole-mount would be omitted or duplicated. Additionally, due to labeled Iba-1+ cells lying outside the immediate focal plane, we analyzed the whole preparation along the Z axis. These procedures were made at 20×, giving an area of 0.1502 mm2 per field analyzed.


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)

Retinal whole-mount. A: areas of retina selected for quantitative analysis of Iba-1+ cells. B: photomicrograph illustrating the method used for arbor-area quantification of Iba-1+ cells. A polygon was drawn manually by connecting the distal-most tips of the Iba-1+ cell processes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4128533&req=5

Figure 1: Retinal whole-mount. A: areas of retina selected for quantitative analysis of Iba-1+ cells. B: photomicrograph illustrating the method used for arbor-area quantification of Iba-1+ cells. A polygon was drawn manually by connecting the distal-most tips of the Iba-1+ cell processes.
Mentions: To determine the effect of OHT on Iba-1+ cells, we quantified these cells in the retinal whole-mounts of naïve (n = 6), contralateral (n = 9), and OHT eyes (n = 9). Twenty-four equivalent areas of the retina were consistently selected for each retinal whole-mount in both the vertical and horizontal meridians which cross the optic nerve (Figure 1A). Each complete meridian selected in the retinal whole-mount was analyzed using the motorized stage of the microscope to scan their whole extension along the X-Y axis, respectively. Thus, all subsequent fields analyzed were contiguous and were examined systematically to ensure that no portion of the retinal whole-mount would be omitted or duplicated. Additionally, due to labeled Iba-1+ cells lying outside the immediate focal plane, we analyzed the whole preparation along the Z axis. These procedures were made at 20×, giving an area of 0.1502 mm2 per field analyzed.

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