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, 41 (5), 127-33

Improved Method of Ink-Gelatin Perfusion for Visualising Rat Retinal Microvessels

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Improved Method of Ink-Gelatin Perfusion for Visualising Rat Retinal Microvessels

Tong Jianbin et al. Acta Histochem Cytochem.

Abstract

To visualize completely rat retinal microvessels, the gelatin-ink perfusion condition was systematically optimized using von Willebrand factor (vWf) immunostaining as control. Whether the vessel showed by the new perfusion condition can be used for double label with neurons or glial cells in the same retina was also tested. Our results showed that infusing rats first with 20 ml of 37 degrees C ink plus 3% gelatin at 140% rat mean arterial pressure (MAP), and subsequently with 20 ml of 37 degrees C ink plus 5% gelatin at 180% rat MAP allowed the ink to completely fill the rat retinal microvessels. Rat retinal microvessels labeled by the perfusion method were more in number than that by vWf immunostaining. Moreover, our data, for the first time, displayed that the improved gelatin-ink perfusion had no effect on and caused no contamination to the following fluorogold labeling or immunostaining of retinal neurons or glial cells in the same tissue. These data suggest that the improved gelatin-ink perfusion technique is a superior method for morphological characterization of rat retinal microvessels, compatible to the double labeling of glial cells and neurons, and it extends the practical scale of the classic method.

Keywords: ink perfusion; microvessel; rat; retina; von Willebrand factor.

Figures

Fig. 1
Fig. 1
Retinal blood vessel labeling with 3% gelatin-ink or 5% gelatin-ink infusion. In the whole-mounted rat retinal specimens that were infused with the 3% gelatin-ink mixture at a perfusion pressure of 140% rat MAP, the peripheral retinal vessels (B) were completely filled, but only partial central vessels (A). In contrast, perfusion conditions of 5% gelatin-ink at a perfusion pressure of 180% rat MAP filled the central retinal vessels (C), but not the peripheral vessels (D). Bar=100 µm.
Fig. 2
Fig. 2
Retinal blood vessels labeling with gelatin-ink infusion or with vWf immunostaining. In the whole-mounted rat retinal specimens that were infused with the gelatin-ink mixture, the central (A) and peripheral (B) vessels were visible as distinct, homogenous, black networks. (C) In rat retinal sections infused with gelatin-ink, black vessels with clear borders were mainly observed in the inner retina. Peripheral vessels (E) labeled by vWf were visible in the whole-mounted retina, while only a few branches were labeled in central vessels (D). The arrow (D) showed the clearly stained small branch of vessels. The number of vessels labeled by vWf (F) in the rat retinal section was obviously lower than that labeled by gelatin-ink infusion (C). The arrow (F) showed the large vessel. Red, vWf immunofluorescence; Blue, Hoechst nuclear staining; NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Bar=100 µm.
Fig. 3
Fig. 3
Double labeling with gelatin-ink infusion and either immunohistochemistry or fluorogold labeling. In the rat retina, NeuN-labeled neurons (A) were observed mainly in the ganglion cell layer, parvalbumin immunoreactivity (B) was observed in the retinal inner layer, and GFAP immunoreactivity (C) was present in glial cell processes. Retinae infused with gelatin-ink resulted in filled vessels, which appear as black cross-sections (D–F). The immunoreactivity of NeuN (D), parvalbumin (E) and GFAP (F) were similar to those of retinae without gelatin-ink infusion. Double labeling of gelatin-ink infusion with fluorogold in the central (G) and peripheral (H) in the whole mount of rat retinae show good contrast between vessels (black) and retinal ganglion cells (yellow). Black: vessel sections perfused with gelatin-ink. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Bar=40 µm.

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