Immunohistochemical assessment of constitutive and inducible heat-shock protein 70 and ubiquitin in human cerebellum and caudate nucleus

Abstract

The distributions of constitutive and inducible 70-kDa heatshock proteins (Hsc70 and Hsp70, respectively) and ubiquitin (Ub) were investigated in autopsy specimens from 24 adult human brains. The objectives were to verify that the milder fixation and celloidin embedding applied to those specimens preserved protein immunoreactivity in the tissue sections, even with extended intervals between death and fixation, and to determine the typical pattern of distribution of the proteins in aged human cerebellum and caudate nucleus. To achieve these objectives, the patterns of immunoreactivity in human specimens were compared with those in normal rat brain after three methods of immersion fixation: 1. 1% Formalin; 2. 10% Formalin; 3. Methacarn (a modification of Carnoy's solution). Additionally, some rats were left refrigerated, but unfixed for up to 24 h to mimic the postmortem interval that commonly occurs prior to fixation of human autopsy material. Tissues were embedded in celloidin, sectioned at 100 microns, and the celloidin dissolved to permit immunostaining. Immunoreactivity for all antigens was greatly diminished in the rat brain by fixation in 10% formalin compared to 1% formalin or methacarn. Rat and human brain tissues fixed in the latter two solutions showed similar patterns of low levels of Hsp70 immunostaining in gray matter and other areas where neuronal somata were concentrated, whereas Hsc70 immunostaining was much greater in those same areas. Little Hsc70 or Hsp70 immunoreactivity was detected in the white matter from either source, but immunoblots of human gray and white matter suggested that white matter contained more Hsc70 and Hsp70 than apparent by tissue section immunoreactivity. Ubiquitin immunostaining in rat and human brain showed the same high levels as Hsc70 in gray matter, but unlike Hsc70, was also visible in white matter. These patterns remained the same in rat brains even if fixation was delayed for 24 h. In three human brain specimens, elevated Hsc70 staining, but not Hsp70 or Ub, was found in a ring pattern similar to that described as the ischemic penumbra in experimentally induced brain ischemia. These results indicated that dilute formalin preserved Hsc/Hsp70 and Ub antigenicity well, and that the proteins had similar distributions in human and rat brains, despite the extended postmortem delay in fixation of the former. They also suggested that evidence of premortem, localized cellular metabolic stress may be preserved in the postmortem human brain by an alteration in the typical distribution of Hsc70.