A battery of cell- and structure-specific markers for the adult porcine retina

Abstract

The pig is becoming an increasingly used non-primate model in experimental studies of human retinal diseases and disorders. The anatomy, size, and vasculature of the porcine eye and retina closely resemble their human counterparts, which allows for application of standard instrumentation and diagnostics used in the clinic. Despite many reports that demonstrate immunohistochemistry as a useful method for exploring neuropathological changes in the mammalian central nervous system, including the pig, the porcine retina has been sparsely described. Hence, to facilitate further immunohistochemical analysis of the porcine retina, we report on the successful use of a battery of antibodies for staining of paraformaldehyde-fixed cryosectioned retina. The following antibodies were evaluated for neuronal cells and structures: recoverin (cones and rods), Rho4D2 (rods), transducin-gamma (cones), ROM-1 (photoreceptor outer segments), calbindin (horizontal cells), PKC-alpha (bipolar cells), parvalbumin (amacrine and displaced amacrine cells), and NeuN (ganglion cells and displaced amacrines). For detecting synaptic connections in fiber layers, we used an antibody against synaptobrevin. For detecting retinal pigment epithelium, we studied antibodies against cytokeratin and RPE65, respectively. The glial cell markers used were bFGF (Müller cells and displaced amacrine cells), GFAP (Müller cells and astrocytes), and vimentin (Müller cells). Each staining effect was evaluated with regard to its specificity, sensitivity, and reproducibility in the identification of individual cells, specific cell structures, and fiber layers, respectively. The markers parvalbumin and ROM-1 were tested here for the first time for the porcine retina. All antibodies tested resulted in specific staining of high quality. In conclusion, all immunohistochemical protocols presented here will be applicable in fixed, cryosectioned pig retina.

Figure 1

(A,A') Photomicrographs show hematoxylin–eosin (H&E)-stained porcine retina. (A) Counterstaining clearly reveals layering of the retina, including cell nuclei and fiber layers, as well as the retinal pigment epithelium, choroid, and sclera. The sclera (connective tissue sheath) and choroid (blood vessels and capillaries) are support layers for the eye. The cell layers are the outer nuclear layer (cones and rods), inner nuclear layers (bipolar, Müller, amacrine, and horizontal cells), and the ganglion cell layer (ganglion cells and displaced amacrine cells). The retinal pigment epithelium consists of pigmented epithelial cells. (A') Seen at a higher-power magnification, the nuclear layers as well as the inner and outer segments of the photoreceptors can be visualized more clearly. GCL, ganglion cell layer; HC, horizontal cell; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segment; NF, nerve fiber layer; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segment; RGC, retinal ganglion cells; RPE, retinal pigment epithelium.

Figure 2

Cones and rods were identified using fluorescence immunohistochemistry with antibodies against recoverin, Rho4D2, transducin, and ROM-1. (A,A′) Recoverin was expressed by rods and cones but with more intensely stained cones found in the location of the retina. Nuclei, processes, and synaptic terminals in the OPL as well as the inner segments could be detected by recoverin labeling. Inset, high-power magnification reveals cell morphology. (B,B′) Rods were specifically labeled with an antibody against Rho4D2. Rho4D2 was found dispersed in the cell cytoplasm, revealing cell nuclei in the ONL and in both the inner and the outer segments. Rho4D2-labeled nuclei were found scattered in the ONL (see higher-power magnification for detail). (C,C′) Transducin was used as a specific marker for cones. Immunostaining visualized weakly labeled nuclei lining the OLM and more strongly labeled ISs and OSs. Moreover, synapses of the cones' processes in the OPL were also clearly visualized (see higher-power magnification for detail). (D) An antibody against the ROM-1 disc protein was utilized to label photoreceptor outer segments. ROM-1 immunostaining was located in the outer segments (see inset for higher-power magnification). Abbreviations as in Figure 1 legend.

Figure 3

Fluorescence immunostaining of the major interneurons of the retina, i.e., horizontal, bipolar, and amacrine cells. In addition to ganglion cell labeling, the output neurons of the retina are shown. (A,A′) Horizontal cells found as a string of pearls located on the scleral side of the INL were detected using an antibody against calbindin. The calbindin-positive cell displayed an elongated cell body with associated bipolar processes running in parallel with the retinal layering. (B,B′) Bipolar cells were identified by PKC-α expression. PKC-α-positive bipolar cell bodies were located close to the OPL, and PKC-α was expressed throughout the cell including the processes and synaptic terminals in the OPL and IPL. (C,C′) Amacrine cell bodies placed in the INL and displaced amacrine cells situated in the GCL were detected using an antibody against parvalbumin. In addition, two parvalbumin-positive parallel fiber layers were visualized in the IPL. (D,D′) RGCs were revealed using an antibody against NeuN. NeuN expression was concentrated in the nucleus and nucleoli of the cells. NeuN was also weakly expressed by displaced amacrine cells in the GCL. Abbreviations as in Figure 1 legend. Scale for left and right columns is shown in A and A′, respectively.

Figure 4

(A) The fiber layers in the OPL and INL were detected using fluorescent immunohistochemistry and the marker synaptobrevin. One fiber layer was noted in the OPL, whereas two layers could be distinguished in the IPL. (B,C) The RPE was identified by using antibodies against RPE65 and cytokeratin. Both markers enabled visualization of the chain-like pattern of cells in the RPE. (B) RPE65 immunostaining clearly detected the dense connected RPE cells, and a very similar staining pattern of the RPE was revealed using an antibody against the Ca²⁺-binding protein cytokeratin (C). Abbreviations as in Figure 1 legend.

Figure 5

Fluorescent immunolabeling for retinal glial cells is shown. (A) Müller cell bodies were visualized using an antibody against bFGF, which intensely stained cell bodies in the INL. bFGF is also known to be expressed by displaced amacrine cells in the GCL, which was also shown here. Application of antibodies against the intermediate filaments, i.e., GFAP and vimentin, resulted in detection of Müller cell processes (B,C). Both of the markers visualized Müller cell fibers reaching from the NF layer into the outer retinal layers. GFAP also labeled astrocytes, primarily located in the GCL and NFL but occasionally also found in the IPL and INL (as shown in Figure 4B). GFAP-labeled astrocytes were often found associated with blood vessels and displayed small cell bodies and multiple short processes. Expression of GFAP was limited to Müller end-feet and/or astrocytes in NF layers and only short thick processes observed in the IPL. Vimentin immunoreactivity was detectable from NFL to IPL/INL with legible processes (C). Abbreviations as in Figure 1 legend.