Patomorfološke i imunohistohemijske karakteristike promena u mozgu starih pasa / Pathomorphological and immunohistochemical characteristics of changes in the brain of aged dogs

Aging is a universal biological process accompanied with numerous changes in the brain. Some of the age-related changes are macroscopically visible, while most of them can be detected only by microscopic examination of the tissues. In this study the presence of age-related changes in the brain of thirty-six adult dogs were examined. These dogs were divided into 2 groups: experimental and control. The control group consisted of 6 dogs aged up to five years while the experimental group comprised 30 dogs older than ten years of age. Necropsies had been performed on all dogs and the brains were macroscopically analyzed, fixed in formalin and embedded in paraffin blocks. The tissue sections were stained by the following methods: hematoxylin-eosin, Congo red, modified Ziehl Neelsen, PAS and immunohistochemical method streptavidin-biotin (LSAB2). As primary antibodies were used: polyclonal antibody against Aβ-14, monoclonal antibody against Aβ1-42, polyclonal antibody against amyloid precursor protein, ubiquitin monoclonal antibody, monoclonal antibody against mеtаlоtiоnеin I and II, GFAP polyclonal antibody and a anti-Tau polyclonal antibody. The most common macroscopic changes in the brain of experimental animals were diffuse leptomeningeal thickening, cortical atrophy with narrowing of the gyri and widening of the sulci, and mild lateral ventricle enlargement. Meningeal fibrosis and fibrosis of the brain blood vessel wall, neuronophagia, satellitosis, astrocytosis, astrogliosis, accumulation of amyloid and lipofuscin and the presence of ubiquitinated and polyglucosan bodies were detected microscopically. The presence of age-related changes was studied in the frontal and parietal cortex, hippocampus, cerebellum and medulla oblongata. The hippocampus, frontal and parietal cortex were the most commonly affected by the above mentioned changes. Amyloid deposits were identified in the walls of cerebral blood vessels and brain parenchyma using Congo red and immunohistochemical staining. The deposits of amyloid in parenchymal and meningeal blood vessels that had been detected by Congo red staining showed a yellow-green fluorescence under polarized light and were Aβ1-14 and Aβ1-42 immunohistochemically positive. Amyloid accumulated extracellularly in the brain parenchyma in the form of plaques and intracellularly. The plaques were Aβ1-14 and Aβ1-42 immunohistochemically positive, while the intracellular deposits were only Aβ1-14 immunohistochemically positive. In old dogs compared to the control group an increase of amyloid precursor protein expression was found. In addition, we observed in old dogs an increase of lipofuscin accumulation in neurons, as well as greater numbers of lipofuscin containing cells. Accumulations of lipofuscin were observed in all examined segments of the dog brains from the experimental group and they were most frequently present in the medulla oblongata. The presence of phosphorylated tau protein was not detected in dog neurons. The greater amount of ubiquitin in glial cells and neurons of old dogs suggests that these cells have increased protein degradation and development of regressive changes. GFAP and metallothionein I and II immunhistochemically positive reactions indicate increased astrocytes reactivity, whereby the reaction was more intense in old dogs. The extracellular and intracellular presence of polyglucosan bodies in the brain tissue of dogs from the experimental group was observed using the PAS method. The observed correlations between changes occurring in the brain of old dogs and those that occur in old people and patients with certain neurodegenerative diseases, indicate that dogs could be used as an animal model for the study of age-related changes in the brain