Objective    To improve the ink perfusion method

thereby enabling it to more completely show the rat retinal microvessels.     Methods    The present study included two parts. The first part comprised of 72 rats that were randomly divided into four groups: only ink group (n=18)

1% gelatin-ink group (n=18)

3% gelatin-ink group (n=18)

and 5% gelatin-ink group (n=18). The animals of each group were subdivided into 90

115

140

165

190 and 215 mmHg subgroups

chosen based on their mean arterial pressure. The ink perfusion was performed via the ascending aorta

and rat retinal microvessels were subsequently detected in whole-mount retinas and retinal sections. The second part was based on the results of the first part

in which 6 rats were infused sequentially with 20 ml ink plus 3% gelatin and then 20 ml ink plus 5% gelatin. The rat retinal microvessels were detected as that in the first part.     Results    Under the perfusion condition that is 40 ml of 37 ℃ ink plus 3% gelatin and 165 mmHg perfusion pressure

the vessels of the peripheral retina were completely shown

whereas only part of the central retinal vessels was shown. In contrast

the perfusion conditions that is 40 ml of 37 ℃ ink plus 5% gelatin and 215 mmHg perfusion pressure

resulted in the central retinal vessels shown very well

but not the peripheral retinal ones. However

when the animals were first perfused with 20 ml of 37 ℃ ink plus 3% gelatin at 165 mmHg perfusion pressure

and subsequently perfused with 20 ml of 37 ℃ ink plus 5% gelatin at 215 mmHg perfusion pressure

both the central retinal vessels and the peripheral retinal vessels were shown very well

without ink leakage in the retina. In addition

the contrast between vessels and surrounding tissues was distinctly improved.     Conclusion    Perfusing animals with 20 ml of 37 ℃ ink plus 3% gelatin at 165 mmHg perfusion pressure

and subsequently with 20 ml of 37 ℃ ink plus 5% gelatin at 215 mmHg perfusion pressure

is hereby demonstrated as good method for morphological characterization of rat retinal microvessels.