Fractal analysis of senile plaque observed in various animal species.

In the present study, the fractal dimension (FD), a concept to determine morphological complexity, was applied to morphological estimation of animal and human senile plaque using a computer-aided method. The FDs of mature plaque in a 17-year-old dog were significantly higher than those of diffuse plaque in 11- to 16-year-old dogs. In both types of plaque, the FD tended to increase as the size expanded and there was a significant difference between the slope values of the approximate line for diffuse and mature plaque. In humans, there was also a significant difference in FD value between diffuse and mature plaque. No significant differences were observed between the two types of plaque in a bear or a cynomolgus monkey. The FD of feline diffuse plaque was significantly lower than that of a camel, bear and monkey. These results indicated that the diffuse and mature plaque of the dog might form in a different manner, and similar events may occur in human senile plaque formation. In addition, specific shapes and different FD values of the diffuse plaque among animals suggested that the original conditions for plaque formation would be different.

Teratologic studies on rat perinates and offspring from dams treated with ethylnitrosourea (ENU).

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues, limbs and male reproductive organs. Recently we clarified that excess cell death caused by apoptosis occurred in these organs and tissues of rat fetuses from dams treated with ENU at day 13 of gestation (GD13). In this study, we examined fetuses at GD21 and offspring at 10 weeks of age after ENU administration to pregnant rats at GD13 in order to clarify the relationship between ENU-induced apoptosis in the fetal tissues and teratogenicity of ENU. Severe intrauterine growth retardation was observed in the ENU group, and the body weight of the offspring in the ENU group was significantly lower than that of the control group throughout the experiment. In addition, a high incidence of microencephaly, ectrodactyly and curved caudal vertebrae was observed in the offspring from dams treated with ENU at GD13. Judging from the results of our previous and present studies, it was strongly suggested that ENU-induced apoptosis in rat fetal tissues may play an important role in the induction of anomalies in the corresponding tissues.

Abnormal neuronal and glial argyrophilic fibrillary structures in the brain of an aged albino cynomolgus monkey (Macaca fascicularis).

An aged albino male cynomolgus monkey (Macaca fascicularis) more than 35 years old died after showing neurological signs including gait disturbance, trembling, drowsing tendency and a decrease in activity. Neuropathological examination revealed glial fibrillary tangles (GFTs) mainly distributed in the putamen, caudate nucleus, thalamic nuclei, substantia nigra, red nucleus, globus pallidus, trapezoid body, pyramid, pons and medulla oblongata of the brain, and neurofibrillary tangles (NFTs) in the thalamic nuclei. These structures were positively stained by the modified Gallyas-Braak (GB) method and immunostained for tau. The tau-positive argyrophilic GFTs were morphologically classified into four types, as in human cases, i.e., tufts of abnormal fibers (TAFs), thornshaped astrocytes (TSAs), glial coiled bodies (GCBs) and argyrophilic threads (ATs) depending on their GB profiles, and GCBs were the major structures in this case. Some of these structures were also immunoreactive for alpha-synuclein. The glial cells possessing the structures were negative for glial fibrillary acidic protein, a marker for astrocytes, indicating that the argyrophilic GFTs were present in oligodendroglia. In addition, marked neuronal loss and ubiquitin-positive spheroid bodies were observed in the substantia nigra and globus pallidus. According to the characteristic distribution of the argyrophilic structures in neurons and glial cells as well as clinical signs, the monkey might have suffered from a neurodegenerative disease such as progressive supranuclear palsy (PSP). This is the first report of the occurrence of a neurodegenerative disease in a nonhuman animal.

Lectin histochemistry in the aged dog brain.

Formalin-fixed and paraffin-embedded canine brains were examined histochemically using 15 selected lectins. Concanavalin A (Con A), Lens culinaris agglutinin, Lycopersicon esculentum agglutinin (LEL) and Limulus polyphemus agglutinin (LPA) labeled neurons in an age-dependent manner. These and some other lectins [Dolichos biflorus agglutinin (DBA), Vicia villosa agglutinin (VVA), Ricinus communis agglutinin 120 (RCA-I), Bandeiraea simplicifolia agglutinin (BSL-I), and Phaseolus vulagaris agglutinin-L (PHA-L)] also age-dependently labeled glial cells. These results indicate that monosaccharide composition and biochemical metabolism in brain cells change with age and that these lectins may be useful as histochemical markers for investigating senile changes in the canine brain. However, no significant correlation was found between ApopTag-positive and lectin-positive cells. Amyloid plaques were positive for Con A, DBA, Glycine maximus agglutinin (SBA), LEL, PHA-L, Limax flavus agglutinin (LFA) and VVA. Among these lectins, VVA, SBA and LFA intensely stained amyloid both in blood vessel walls and senile plaque cores. Therefore, the sugar residues recognized by these lectins likely play specific roles in beta-amyloid deposition in the aged dog brain.

Deposition of amyloid beta protein (A beta) subtypes [A beta 40 and A beta 42(43)] in canine senile plaques and cerebral amyloid angiopathy.

To clarify the immunohistochemical features of canine senile plaques (SPs) and cerebral amyloid angiopathy (CAA), the distribution of the amyloid beta protein (A beta) subtypes A beta 40 and A beta 42(43), A beta precursor protein (APP), and glial cell reaction were examined in the brains of seven aged dogs (12-18 years). A beta 42(43) was found to be deposited in all types of SPs, whereas A beta 40 was deposited only in mature (classical and primitive) plaques. CAA, which was located along parenchymal and meningeal arterioles and capillaries, consisted of both subtypes of A beta. APP was exhibited in normal and degenerative neurons and swollen neurites of mature plaques. It was, therefore, considered that A beta 42(43) in diffuse plaques might be derived from APP in neurons, while A beta 40 and A beta 42(43) in mature plaques might be generated from APP in swollen neurites in the plaque. In contrast to the case in humans, in whom deposition of A beta 40 and A beta 42(43) in the mature plaques is predominantly associated with microglial reaction, in dogs we found that it was closely associated with astroglial reaction. The present findings showed characteristics of canine SPs which are different from those of humans.

Age-related changes in susceptibility of mice to low-virulent mouse hepatitis virus (MHV-2-CC) infection.

This study was performed to examine mouse age-dependent changes in susceptibility to MHV-2-CC-infection and participation of macrophages in such changes in BALB/c mice. One-week-old mice were fully susceptible (mortality, 100%), 2-week-old semi-susceptible (36%), and 3- and 4-week-old fully resistant (0%) to MHV-2-CC, respectively. Such age-dependent differences corresponded well with the differences in the virus titers in the liver, spleen and blood and in the severity of liver lesions. In 1-week-old mice with peritoneal exudate cells (PEC) transferred from 4-week-old mice and infected with MHV-2-CC, a slight prolongation of survival time was recorded, although there was no difference in mortality. In 3-week-old mice infected with MHV-2-CC after silica-treatment to suppress macrophages, there was no significant change in susceptibility. In macrophages infected with MHV-2-CC in vitro, the virus replicated better in macrophages obtained from younger mice. These results suggest that macrophages may play a small role in the age-related development of resistance to MHV-2-CC infection in BALB/c mice.

T-2 toxin-induced apoptosis in lymphoid organs of mice.

Lymphoid organs of male and female mice of 4 strains (ICR: CD-1, BALB/c, C57BL/6 and DBA/2) were histologically and biochemically examined at 24 hours after oral inoculation of T-2 toxin (0, 2.5, 5 and 10 mg/kg b.w.). Light microscopically, dose-dependent decrease in number of lymphocytes was observed in the thymic cortex and splenic follicles. The nuclei of lymphocytes showed pyknosis or karyorrhexis, and they were positively stained by the modified TUNEL method which detects fragmented DNA in situ. Electron microscopic characteristics of damaged lymphocytes were shrinkage of the cell body, nuclear chromatin condensation and fragmentation. Agarose gel electrophoresis of DNA extracted from the thymus showed DNA fragmentation into nucleosome units, i.e. ladder formation. The above-mentioned findings clearly showed that T-2 toxin could induce apoptotic cell death in the lymphoid organs of mice. These changes were more prominent in female BALB/c and C57BL/6 mice.

Apoptosis in the aged dog brain.

Apoptosis similar to that seen in Alzheimer's disease patients was found in the brain of aged dogs by the TUNEL method of detecting in situ DNA fragmentation. Apoptosis was observed in both neurons and glial cells, and was morphologically characterized by round and swollen cytoplasm and aggregated nuclear chromatin, although these changes were slight. Neurons and astrocytes in the gray matter and oligodendrocytes in the white matter were affected. The number of ApopTag-positive brain cells increased slightly with age, but was not correlated to the number of senile plaques. A good correlation between the number of ApopTag-positive cells and the dementia index was clearly found. The present study indicates that brain cell apoptosis could account for dementia in aged dogs and suggested that aged dogs may be useful as a simplified animal model for Alzheimer's disease in man.

Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys.

In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (alpha ACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular A beta, apoE, and alpha ACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular A beta and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.

Immunohistochemical study on the deposition of apolipoprotein E in cerebral and islet amyloidoses in cynomolgus monkeys (Macaca fascicularis).

To investigate the role of apolipoprotein E (apo E) in amyloidoses of cynomolgus monkeys, the localization of apo E in cerebral amyloid, including senile plaques and cerebrovascular amyloid, and in islet amyloid was examined immunohistochemically. Mature types of senile plaques with amyloid deposits and cerebrovascular amyloid showed intense immunoreactivity to both antisera to apo E and amyloid beta protein (A beta). In contrast, diffuse plaques without obvious Congophilic amyloid showed weak immunoreactivity to antiserum to apo E, but intense reactivity to antiserum to A beta. In addition, the number of these apo E-positive diffuse plaques was small compared with that of A beta-positive plaques. On the other hand, diabetic islet amyloid that was negative with A beta, reacted intensely with antiserum to apo E. These findings suggest that apo E plays an important role in amyloid fibril formation in several types of amyloidoses.

Senile plaques in very aged cats.

Senile plaques were found in the cerebral cortices of three very aged cats (more than 18 years old). The plaques consisted of a coarse assembly of silver staining-positive materials, and was morphologically different from the well-known classical, primitive, and diffuse plaques. Congophilic amyloid angiopathy was observed in a few cortical arterioles of the oldest cat (20 years old). The senile plaques and a few cortical blood vessels were immunopositive for amyloid beta-protein (A beta). A beta-positive materials were also sparsely distributed in the cortical neuropil but did not form senile plaques there. These findings should help to clarify the development of senile plaques and the early stage of A beta deposition.