Figure 1.
Rod-like microglia were restricted to the NFL of OHT-eyes.
Iba-1+ cells in the NFL and RGC layer. Retinal whole-mounts. In naïve (A) and contralateral (B) eyes, Iba-1+ cells were distributed in a mosaic of tiled cells that built networks throughout the entire retina. In OHT-eyes (C), Iba-1+ cells aligned forming parallel trains (arrows) composed by multiple cells exhibiting rod-like morphology. The trains were more evident in the central retina. (NFL: nerve-fibre layer; RGC: retinal ganglion-cell layer; OD: optic disc; OHT: ocular hypertension).
Figure 2.
Rod-like microglia did not relate to retinal blood vessels or astrocytes.
Iba-1+ and GFAP+ cells in the NFL and RGC layer. Retinal whole-mounts. In naïve (A,D) and contralateral (B,E) eyes, most Iba-1+ cells had a ramified morphology (A1, B1, D1, E1: arrowhead) and their somas were located on or near the vessel walls (A3, B3, D3, E3: asterisks). GFAP+ astrocytes were located among ramified Iba-1+ cells and like these, were related to the vessels (A2-A3, B2-B3, D2-D3, E2-E3). In OHT-eyes (C,F), astrocytes and Müller cells were reactive (C2-C3, F2-F3). No alignment or co-localization of GFAP+ astrocytes with rod-microglia (C1, F1: arrows) was detected. Trains of rod-like microglia were more evident in the central (C1, C3) than in the peripheral retina (F1, F3). (NFL: nerve-fibre layer; RGC: retinal ganglion-cell layer; OHT: ocular hypertension). .
Figure 3.
MHC-II was upregulated in contralateral and OHT-eyes.
Iba-1+ and MHC-II+ cells in the NFL and RGC layer. Retinal whole-mounts. In naïve (A1-A3), contralateral (B1-B3), and OHT (C1-C3) eyes, some Iba-1+ cells displayed bipolar morphology and perivascular arrangement (arrowhead). These cells were observed mainly in the large retinal vessels (in the vicinity of the optic nerve) and in the collecting tube (in the peripheral retina) and expressed MHC-II. This MHC-II expression, constitutive in naïve eyes (A2-A3), was upregulated in contralateral (B2-B3) and OHT-eyes (C2-C3). (NFL: nerve-fibre layer; RGC: retinal ganglion-cell layer; OHT: ocular hypertension; asterisk: blood vessel; arrow: rod-microglia).
Figure 4.
Rod-like microglia expressed MHC-II and related to each other in three different patterns.
Iba-1+ and MHC-II+ cells in the NFL and RGC layer. Retinal whole-mounts. Rod-like microglia expressed MHC-II (A2-A3, B2-B3, C2-C3). In the trains, they related to each other in three ways. Process-process (A1-A3): processes of neighbouring rod-microglia in the trains seem to be intertwined; process-soma (B1-B3): the process of one rod-like microglia seems to surround the cell body of the next rod-like microglia in the train; soma-soma (C1-C3): two cells bodies are in apparent close contact. (NFL: nerve-fibre layer; RGC: retinal ganglion-cell layer; OHT: ocular hypertension; asterisk: soma; arrow: processes).
Figure 5.
The processes of rod-microglia penetrates the IPL.
Iba-1 and NF-200 immunostaining. Retinal whole-mounts. Using the z stack tool associated with the microscope, we observed that some processes of rod-microglia penetrate the ganglion-cell layer and inner plexiform layer (A). The pressure observed by the cover slip on the whole-mount caused retinal-like section effect on one edge of the tissue that revealed that, in the IPL, rod-microglia were apparently in close relation with the dendrites of NF-200+RGCs (B1,B3). (INL: inner plexiform layer; IPL: inner plexiform layer; GCL: ganglion-cell layer; NFL: nerve-fibre layer; RGC: retinal ganglion-cell layer; asterisk: soma; arrow: rod-microglia processes).
Figure 6.
Rod-like microglia did not take either CD86 or Ym1 staining.
Iba-1, CD86, MHC-II and Ym1 immunostaining in the NFL. Retinal whole-mounts. In OHT-eyes (A1-A3, C1-C3) CD86 immunostaining (A2, A3) and Ym1 immunostaining (C2-C3: inset) was restricted to few globular cells (arrowhead) located in the NFL and in the vitreal surface of the retina. Rod-like microglia (arrow) did not show either CD86 or Ym1 immunostaining. In contralateral eyes (B1-B3, D1-D3) no CD86+ or Ym1+ cells were detected. (NFL: nerve-fibre layer).
Figure 7.
Rod-microglia had different stages of CD68 staining.
Iba-1+ and CD68+ cells. Retinal whole-mounts. With respect both to morphological features and to CD68 immunostaining patterns, three stages of rod-like microglia were detected: (A1-A3) rod-like microglia with elongated cell bodies and processes prominently projected from the poles and an intense punctate CD68 staining (arrowhead); (B1-B3) cells with shorter and thicker processes, and coarse patches of CD68 immunoreaction (arrow); (C1-C3) fusiform cell bodies with short and thick bipolar processes and intense CD68 positive cytoplasm (asterisk). In all cases, CD68 immunoreaction was found both in the cytoplasm and processes.
Figure 8.
Rod-like microglia was related to axons.
Iba-1 and NF-200 immunostaining. Retinal whole-mounts. In naïve (A) and in contralateral eyes (B) the ramified microglia (arrowhead) sent some processes to the axons but did not run parallel to the axons. By contrast, the rod-microglia trains in OHT-eyes (C) run parallel to and close to axons (arrow). (OHT: ocular hypertension).
Figure 9.
Rod-microglia sent processes to NF-200+RGCs somas and dendrites.
Iba-1 and NF-200 immunostaining. Retinal whole-mounts. In OHT-eyes there was an abnormal NF-200 staining of the soma and primary dendrites of some RGCs (A2-C2). Rod-microglial run parallel to and close to axons, with minimal gaps between the two (empty arrowhead). Rod-microglia sent processes to NF-200+RGCs bodies and dendrites (arrows in A1-B2). The processes of the rod-microglia were apparently in close contact with NF-200+RGCs somas and dendrites (empty arrow in inset). In some instances rod-microglia deviates from its straight parallel to the axon and, with its processes, surrounds the soma and proximal dendrites of NF-200+RGCs (arrowhead in C1, C2).
Figure 10.
Rod-like microglia were absent in the scleral lasered group of eyes.
Iba-1+ cells in the NFL and RGC layer. Retinal whole-mounts. In eyes receiving laser in the non-draining portion of the sclera, lasered (A) as well as contralateral untreated eyes (B) Iba-1+ cells were distributed in a mosaic of tiled cells that built networks throughout the entire retina. No rod-like microglia was detected in the NFL of this group of animals (NFL: nerve-fibre layer; RGC: retinal ganglion-cell layer; OD: optic disc).
Figure 11.
Macroglial MHC-II expression was upregulated in the scleral lasered group of eyes.
Iba-1, GFAP and MHC-II immunostaining. Retinal whole-mounts. Both in lasered (A1-A4) and contralateral untreated (B1-B4) eyes, MHC-II expression was upregulated in astrocytes (arrow) and Müller cells (empty arrowhead). Some microglia in the NFL (arrowhead) had a constitutive MHC-II expression. (NFL: nerve-fibre layer).