Effects of anoxia on serum immunoglobulin and albumin leakage through blood-brain barrier in mouse cerebellum as revealed by cryotechniques.

The purpose of this study was to examine time-dependent topographical changes of leaking proteins from blood vessels in the mouse cerebellum to assess the effect of normal blood circulation on the blood-brain barrier (BBB). The distribution of leaking serum proteins was immunohistochemically examined by various cryotechniques including our "in vivo cryotechnique". The cryofixed cerebellar tissues were processed for the freeze-substitution method, and finally embedded in the common paraffin wax. Serial de-paraffinized sections were immunostained by anti-mouse immunoglobulin-G (IgG) or albumin antibody. By combination of the "in vivo cryotechnique", in which normal blood flow into the cerebellum was always kept in vivo, with the freeze-substitution method, serum IgG and albumin were clearly localized inside of cerebellar blood vessels. To examine abnormal leakage of blood vessels as a model of anoxia, some cerebellar tissues were partially removed from brains in the mouse skull and quickly frozen in the isopentane-propane within a minute. In such resected cerebellar tissues, serum IgG and albumin were diffusely immunostained in large areas around the blood capillaries, probably because of easy leakage of the serum components through the immediately changed BBB. To the contrary, no serum protein could be identified outside blood capillaries under living conditions of the anesthetized mice. The present combination method, both "in vivo cryotechnique" and freeze-substitution, for immunohistochemistry enabled us to examine the in vivo localization of serum components in mouse brains due to alteration of the BBB.