Immunohistochemical study of pig retinal development

Abstract

Purpose: The pig eye is similar to the human eye in terms of anatomy, vasculature, and photoreceptor distribution, and therefore provides an attractive animal model for research into retinal disease. The purpose of this study was to characterize retinal histology in the developing and mature pig retina using antibodies to well established retinal cell markers commonly used in rodents.

Methods: Eyes were enucleated from fetuses in the 9th week of gestation, 1 week old piglets and 6 months old adult animals. Eyeglobes were fixed and cryosectioned. A panel of antibodies to well established retinal markers was employed for immunohistochemistry. Fluorescently labeled secondary antibodies were used for signal detection, and images were acquired by confocal microscopy. Mouse retina at postnatal day (P) 5 was used as a reference for this study to compare progression of histogenesis. Most of the primary antibodies have previously been used on mouse tissue.

Results: Most of the studied markers were detected in midgestation pig retina, and the majority had a similar distribution in pig as in P5 mouse retina. However, rhodopsin immunolabeling was detected in pig retina at midgestation but not in P5 mouse retina. Contrary to findings in all rodents, horizontal cells were Islet1-positive and cones were calbindin-immunoreactive in pig retina, as has also been shown for the primate retina. Recoverin and rhodopsin immunolabeling revealed an increase in the length of photoreceptor segments in 6 months, compared to 1 week old animals.

Conclusions: Comparison with the published data on human retina revealed similar marker distribution and histogenesis progression in the pig and human retina, supporting the pig as a valuable animal model for studies on retinal disease and repair. Furthermore, this study provides information about the dynamics of retinal histogenesis in the pig and validates a panel of antibodies that reliably detects developing and mature retinal cell phenotypes in the pig retina.

Figure 1

Pig retinal cryosections labeled immunohistochemicaly for nestin, Pax6 and Ki67. A: Nestin immunoreactivity is evident throughout GW9 retina. B: Nestin positive fibers are in the GCL and IPL (arrows) in 1W retina. C: Cells in the GCL, differentiating amacrine (thick arrows), horizontal (arrowheads) and putative retinal progenitors (thin arrows) are Pax6-positive in GW9 retina. D: Immunoreactivity for the mitotic marker Ki67 in GW9 retina reveals distribution of progenitor cells similar to that labeled for Pax6 (thin arrows). E: Cells in the GCL (thick arrows), amacrine (thin arrows) and some horizontal cells (arrowheads) are Pax6-positive in 1W retina. Nuclei are labeled with PI (red).

Figure 2

P5 mouse retinal sections labeled immunohistochemicaly. A: Radial processes are positive for nestin. B: Ganglion, amacrine and horizontal cells (arrows) are Pax6- positive. C: Ganglion cells are labeled for Brn3a. D: GCL, amacrine cells, IPL, and OPL are NF-160-positive. E: Displaced amacrine and amacrine cells are AP2α-positive. F: GCL, IPL, amacrine cells and OPL are labeled for calretinin. G: Ganglion, amacrine and horizontal cells are calbindin-positive. H: IPL, ganglion, amacrine and horizontal cells (arrowheads) are labeled for GAD65. I: Ganglion, amacrine (arrows) and bipolar cells (arrowheads) are Islet1-positive. J: GCL, IPL, INL and OPL are PKCα-positive. K: ONL and some cells in the GCL (arrows) are positive for recoverin. Neurofiber layer, INL and OPL are labeled for P75^NTR (L) and GS (M). Nuclei are labeled with PI (red).

Figure 3

Pig retinal cryosections labeled immunohistochemicaly for the GCL protein markers. A: Only ganglion cells are labeled for Brn3a in GW9 retina. B: Ganglion cells are Brn3a-positive in 1W retina. C: Cells in the GCL (thick arrows), IPL, some developing amacrine (thin arrows) and horizontal cells (arrowheads) are positive for NF-160 in GW9 retina. D: GCL, neurofiber layer, and processes in IPL and OPL are NF-160-positive in 1W retina. E: Weak labeling for AP2α in the GCL (thick arrows) and prospective INL (thin arrows) is present in GW9 retina F: Cells in the GCL and amacrine cells in the INL are APα2-positive in 1W retina. Nuclei are labeled with PI (red).

Figure 4

Pig retinal cryosections labeled immunohistochemicaly for calretinin, calbindin, and GAD65. A: Ganglion (thick arrows), amacrine (thin arrows) and horizontal cells (arrowheads) are calretinin-labeled in GW9 retina. GCL, IPL, amacrine and horizontal cells are calretinin-positive in 1W retina (B) and 6M (C) retina. Cells in the GCL, amacrine (thick arrows), differentiating horizontal (arrowheads), and putative cones (thin arrows) are calbindin-positive in GW9 retina. GCL, IPL, amacrine, horizontal cells, and cones (thin arrows) are calbindin-positive in 1W (E) and 6M (F) retina. G: GCL, IPL, amacrine (arrows) and horizontal cells (arrowheads) are GAD65-positive in GW9 retina. IPL, cells in the GCL (thick arrows), amacrine cells (thin arrows) and OPL (arrowheads) are GAD65-positive in 1W (H) and 6M (I) retina. Nuclei are labeled with PI (red).

Figure 5

Pig retinal cryosections labeled immunohistochemicaly for Islet1 and PKCα. A: Ganglion (thick arrows), amacrine (thin arrows) and horizontal cells (arrowheads) are Islet1-positive in GW9 retina. B: Ganglion, amacrine (thick arrows), bipolar (thin arrows) and horizontal cells (arrowheads, also labeled with calbindin in inset B) are Islet1-positive in 1W (B) and 6M (C) retina. D: GCL (thick arrows), IPL, prospective INL (thin arrows), and developing OPL (arrowheads) are PKCα-positive in GW9 retina. E: Bipolar cell bodies (arrowheads) and their processes (arrows) are PKCα-positive in 1W retina. F: Bipolar cell bodies (arrowheads) and axonal endings (arrows) are PKCα-labeled in 6M retina. Nuclei are labeled with PI (red).

Figure 6

Pig retinal cryosections labeled immunohistochemicaly for photoreceptor protein markers. A: Cells in the prospective ONL are positive for recoverin (arrows) in GW9 retina. ONL and photoreceptor segments are recoverin-labeled in 1W (B) and 6M (C) retina. D: Some differentiating rod photoreceptors are positive for rhodopsin in GW9 retina (arrows). E: Strong rhodopsin-labeling is present in the outer segments (thick arrows) in 1W retina. Inner segments (arrowheads) and cell bodies (thin arrows) are less intensely stained. F: Rhodopsin-positive inner (arrowheads) and outer (thick arrows) segments are more elongated in 6M compared to 1W retina. Some rod cells bodies are also labeled (thin arrows) in 6M retina. Cone outer segments are labeled for GNAT2 in 1W (G) and 6M (H) retina (thin arrows). Nuclei are labeled with PI (red).

Figure 7

Pig retinal cryosections labeled for glial protein markers. A: Processes in the neurofiber layer, GCL, OPL, inner NBL, and developing OPL are P75^NTR-positive in GW9 retina. Processes in the GCL, IPL, and INL are P75^NTR-positive in 1W (B) and 6M (C) retina. D: Processes in the GCL (thick arrows), NBL (thin arrows), and developing OPL (arrowheads) are labeled for GS in GW9 retina. GS immunolabeling is present in the GCL, IPL, and INL in 1W (E) and 6M (F) retina. G: Immature astrocytes are GFAP-positive in GW9 retina. H: Processes in neurofiber and GCL are GFAP- positive in 1W retina. I: Elongated GFAP-positive processes in 6M retina. J-M: GFAP-immunopositive astrocytes are PKCα-positive, weakly in 1W (J, K, arrows) but strongly in 6M retina (L, M, arrows). N: A Z-stack image shows extensive double GFAP-PKCα labeling at 6M (arrows). X- and Y- planes are shown to bottom and to the right of the image.