Methods in laboratory investigation. Application of test substances to the surface of rat liver in situ: opposite effects of insulin and isoproterenol on cellular autophagy.

Small plastic rings were applied to the surface of intact rat liver and used as microvessels in short-term experiments (10 to 20 minutes) for the local surface application of test substances. Tissue within the ring was then fixed using OsO4 in phosphate buffer. Excellent ultrastructural preservation of hepatocytes and sinusoids, including their contents, was obtained in the superficial tissue layer. By combining this method with quantitative electron microscopy we found that insulin decreased the volume fraction and numerical density of autophagic vacuoles, in comparison with control areas of the same livers exposed to Ringer's solution alone. Isoproterenol had opposite effects. One of the advantages of this method is that counterregulation, which must always be considered following systemic application of test substances, can be excluded as a possible explanation of the effects observed.