Jaime Rofina

Immunohistochemical investigation of the brain of aged dogs. I. Detection of neurofibrillary tangles and of 4-hydroxynonenal protein, an oxidative damage product, in senile plaques.

In the aging dog brain lesions develop spontaneously. They share some morphological characteristics with those of Alzheimers disease in man. Diffuse and primitive plaques are well known, whereas neuritic plaques rarely develop. Neurofibrillary tangles have not been seen in the canine. The aim of the present investigation was to study major age-related changes of the dogs brain using paraffin sections with respect to cross-immunoreactivity oftau, Aβ protein and other immunoreactive components including hydroxynonenal protein, which is a marker for oxidative damage. The occurrence of neurofibrillary tangles and of the protein tau therein was studied in serial brain sections of two dogs with the Gallyas stain and by immunohistochem-istry with three different antibodies against tau. Senile plaques were stained with a monoclonal anti-Aβ (residues 8-17), polyclonal anti-apolipoprotein E and a monoclonal antibody against 4-hydroxynonenal (HNE). Amyloid deposits and controls were screened by Congo red staining viewed in fluorescent light, followed by polarized light for green birefringence. With the Gallyas stain and one of the antisera against tau, neurofibrillary tangles were revealed in a similar dispersed pattern, whereas the other antitau antisera gave negative results. With the anti-HNE a positive reaction was found in cerebral amyloid deposits and in vascular wall areas where amyloid deposition was confirmed by Congo-red staining, and in perivascular cells and in some neurons. These results indicate that the canine with his tangles and plaques which show oxidative changes, forms a spontaneous model for understanding the early changes and their interrelationships in Alzheimers disease.

Cognitive disturbances in old dogs suffering from the canine counterpart of Alzheimer's disease

In geriatric dogs, Alzheimer-like behavior is frequently observed. This behavior has been classified by several authors using questionnaires and a correlation has been described between cognitive dysfunctions and Alzheimer-like pathology. In the present study, cognitive performance was correlated with brain pathology for 30 dogs of varying ages. Within these animals, two age-matched groups of old dogs with and without behavioral changes were compared. The behavioral changes were analyzed and scored with questionnaires and necropsy was performed to rule out any other cause for changed behavior. Measurements, (immuno)-histochemical staining and fluorescence microscopy were used to detect cortex atrophy, amyloid, rest-products of oxidative damage, demyelination and accumulations of macrophages in the brains of these dogs. Spearman rank correlation coefficients (r) were calculated and adjusted according to Bonferonni. In the whole group (young to very old dogs), the age of the animal showed a significant correlation with various behavioral changes (r = 0.7 to 0.9, P < 0.01). The dementia score correlated significantly (r = 0.6 to 0.8, P < 0.01) with all the brain lesions studied, except one, i.e. demyelination (r = -0.4, P > 0.05). These results suggest that a questionnaire can be used to diagnose Alzheimer-like changes in canine practice. Oxidative damage on a cellular and a nuclear level plays an important role in behavior changes.

A mRNA PCR for the diagnosis of feline infectious peritonitis.

Abstract A reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of feline coronavirus (FCoV) messenger RNA in peripheral blood mononuclear cells (PBMCs) is described. The assay is evaluated as a diagnostic test for feline infectious peritonitis (FIP). It is based on a well-documented key event in the development of FIP: the replication of virulent FCoV mutants in monocytes/macrophages. To detect most feline coronavirus field strains, the test was designed to amplify subgenomic mRNA of the highly conserved M gene. The test was applied to 1075 feline blood samples (424 from healthy, 651 from sick cats suspected of FIP) and returned 46% of the diseased cats as positive for feline coronavirus mRNA in their peripheral blood cells; of the healthy cats, 5% tested positive. Of a group of 81 animals in which FIP had been confirmed by post-mortem examination, 75 (93%) tested positive, whereas 17 cats with different pathologies (non-FIP cases) all tested negative. In view of the low rate of false-positive results (high specificity) the mRNA RT-PCR may be a valuable addition to the diagnostic arsenal for FIP.

Histochemical accumulation of oxidative damage products is associated with Alzheimer-like pathology in the canine

An important lesion in Alzheimers disease (AD) patient brains is the neurofibrillary tangle (NFT). Hyperphosphorylated tau is its major component. In a former paper we described some NFT in the canine brain. During aging, moreover, advanced glycation end products (AGE) might accumulate. Glycated tau induces lipid peroxidation in vivo and tau and AGE antigens have been mentioned to co-localize in NFT. This indicates that AGE may play an important role in Alzheimer disease (AD) by oxidation of tau. The aim of the present study was to investigate amyloid, neurofibrillary tangles, Abeta precursor protein, Abeta, tau, ubiquitin, advanced glycation end products, 4-hyroxynonenal protein and lipofuscin in a series of dogs of varying ages. The results showed a significant positive correlation between age and amyloid quantity (Congo red staining), HNE staining and lipofuscin (LF), and between amyloid quantity and HNE staining and LF. Staining for AβPP seemed to have a tendency to increase with age, whereas staining for tau, ubiquitin and AGE each only gave limited positive results in a proportion of the older dogs. Preliminary studies including loss of cognitive capabilities in the older dogs and chemical measurement of lipofuscin-like pigment (LFP) accumulation in brain extracts revealed an increase with old age and dementia. The Congo red, HNE and LF results suggest that deposition of amyloid with aging might be associated with formation of end products of lipid peroxidation. The finding of the limited positive signals for tau, ubiquitin and AGE in some old cases might indicate that the spontaneous brain pathology of the aged dog reveals similarities to early stages observed in AD in humans especially those with Down syndrome.

Canine counterpart of senile dementia of the Alzheimer type: amyloid plaques near capillaries but lack of spatial relationship with activated microglia and macrophages

Senile plaques and cerebrovascular amyloidosis are major histopathological lesions in the brains of aged dogs. Different types of amyloid beta protein (Aβ) positive plaques are known: diffuse ones and neuritic plaques. Diffuse plaques may contain membrane-bound Aβ and/or small amounts of amyloid fibrils. Neuritic plaques are cored plaques with clusters of amyloid fibrils and degenerating neurites. In human amyloid plaques, a pathogenetic role for mcroglia cells has been described. The aim of this investigation was to study microglia cells in relationship to canine plaques and to investigate the localisation of amyloid plaques in relationship to vasculature. The lesions were studied by hematoxylin and eosin Congo red staining and immunohistochemistry with anti-Aβ for plaques, with Mac 387, anti lysozyme and a series of lectins for mononuclear cells, with anti von Willebrand Factor and Lycopersicon esculentum (tomato) lectin for the endothelium of brain capillaries. Diffuse Aβ-positive plaques were found in dogs of 10.8 years and older, and cored Appositive plaques with birefringent amyloid in Congo red-stained sections in subjects of 15 years and older. Accumulation of microglia cells in relationship to the plaques was not obvious. With anti Aβ8–17 the distribution of the plaques in the cortical layers varied. The younger dogs had primarily diffuse plaques in the deeper layers of the cortical grey matter. The older dogs showed more cored plaques than diffuse plaques which were found throughout all cortical grey matter layers. With anti Aβx-42 more plaques were found positive, especially diffuse ones, whereas staining results of anti Aβx-40 were more confined to amyloid plaques and vascular amyloid. A close spatial relationship was found between the cored plaques and capillaries.

Oxidative Stress, Aging, and Central Nervous System Disease in the Canine Model of Human Brain Aging

Decline in cognitive functions that accompany aging in dogs may have a biologic basis, and many of the disorders associated with aging in dogs may be mitigated through dietary modifications that incorporate specific nutraceuticals. Based on previous research and the results of laboratory and clinical studies, antioxidants may be one class of nutraceutical that provides benefits to aged dogs. Brains of aged dogs accumulate oxidative damage to proteins and lipids, which may lead to dysfunction of neuronal cells. The production of free radicals and lack of increase in compensatory antioxidant enzymes may lead to detrimental modifications to important macromolecules within neurons. Reducing oxidative damage through food ingredients rich in a broad spectrum of antioxidants significantly improves, or slows the decline of, learning and memory in aged dogs.

Pathology of the Aging Brain in Domestic and Laboratory Animals, and Animal Models of Human Neurodegenerative Diseases

According to the WHO, the proportion of people over 60 years is increasing and expected to reach 22% of total world’s population in 2050. In parallel, recent animal demographic studies have shown that the life expectancy of pet dogs and cats is increasing. Brain aging is associated not only with molecular and morphological changes but also leads to different degrees of behavioral and cognitive dysfunction. Common age-related brain lesions in humans include brain atrophy, neuronal loss, amyloid plaques, cerebrovascular amyloid angiopathy, vascular mineralization, neurofibrillary tangles, meningeal osseous metaplasia, and accumulation of lipofuscin. In aging humans, the most common neurodegenerative disorder is Alzheimer’s disease (AD), which progressively impairs cognition, behavior, and quality of life. Pathologic changes comparable to the lesions of AD are described in several other animal species, although their clinical significance and effect on cognitive function are poorly documented. This review describes the commonly reported age-associated neurologic lesions in domestic and laboratory animals and the relationship of these lesions to cognitive dysfunction. Also described are the comparative interspecies similarities and differences to AD and other human neurodegenerative diseases including Parkinson’s disease and progressive supranuclear palsy, and the spontaneous and transgenic animal models of these diseases.

Analysis of cDNA sequences of feline SAAs

Feline amyloidosis is an uncommon disorder caused by deposits of amyloid in a variety of organs. Most frequently encountered types are amyloid derived of pancreatic islet amyloid polypeptide (AIAPP) in older cats and of the apolipoprotein, apo-serum amyloid A (AA) in Abyssinian/Somali (Aby) and Siamese/Oriental (Siam) cats occurring at a relatively young age. For the AA protein of the Aby, Siam and domestic shorthair cat (DSH) breed different amino acid sequences have been described. It is not yet clear, however, whether the tendency to develop AA amyloidosis in Aby and Siam is associated with specific apoSAA protein sequences and whether this is breed specific. In this study, DNA from one Siam and two DSH cats revealed on Southern blot three bands suggesting at least three genes or gene clusters. The SAA cDNAs of hepatic mRNA from three Abys, five Siams and five DSHs were amplified by RT-PCR, cloned and sequenced. Siams and Abys had limited SAA sequence variability. All five Siams, three of which were positive for amyloid, had the amyloidogenic Siam SAA and the amyloidogenic Aby SAA sequence. Two of the Abys, both with amyloid, had the amyloidogenic Aby SAA sequence. The third Aby, without amyloid, missed its amyloidogenic sequence. The SAA sequences of the DSHs found in the present preliminary survey, suggested a possible tendency for more variability, whereas the amyloidogenic Siam as well as the amyloidogenic Aby sequence were found once. Up to five different sequences were found in a single animal. All five DSHs, moreover, had a specific sequence lacking in the Siams and Abys. The present results, especially those of the Siams, favor that in addition to the occurrence of amyloid associated SAA genes other factors such as infections and inflammatory processes are involved in the development of phenotypical amyloidosis.