Lipofuscin Pigment Research Articles

Neuropathology of Alzheimer's disease: what is new since A. Alzheimer?

Alzheimer's disease results from severe cytoskeletal alterations in only a few neuronal types within the human central nervous system. These intraneuronal changes take the form of neurofibrillary tangles and neuropil threads. Beginning in predisposed induction sites in the allocortex, the lesions follow a predictable sequence as they engulf other territories of the cerebral cortex and a specific set of subcortical nuclei. Some components of the brain are devastated, while others remain intact until the end phase of the disease. Assessment of the location of the afflicted neurons and the severity of the lesions allows the distinction of stages in the development of the disease. The degenerative process begins with the emergence of the first lesions, at whatever age it occurs. The illness remains subclinical for years, and proceeds inexorably, gradually laying waste to higher order limbic system centers. Clinical symptoms are observed only late in the course of the disease, and their appearance is usually concurrent with the encroachment of the destructive process upon neocortical association areas. The sequence of destruction bears a striking resemblance to the inverse sequence of cortical myelination. Late myelinating areas and layers develop the disease-related changes earlier and at higher densities than those which are myelinated early. The brain of the human adult is heavily laden with intraneuronal deposits of lipofuscin and neuromelanin pigment. The average density of neuronal pigmentation in given cortical areas mirrors the density of cytoskeletal lesions that develop in the course of the disease. Pigment-laden neuronal types giving rise to a single long, thin, unmyelinated or sparsely myelinated axon are particularly prone to developing Alzheimer's disease-related cytoskeletal changes.

HISTOCHEMISTRY AND ULTRASTRUCTURE OF THE CRYPT CELLS IN THE DIGESTIVE GLAND OF APLYSIA PUNCTATA (CUVIER, 1803)

The crypt cells lining the Aplysia punctata digestive tubules comprise of three types of cell; calcium, excretory, and thin cells. The calcium cells play a role in osmoregulation, mineral storage, exocrine secretion, iron detoxification, and excretion processes. They possess well- developed microvilli and a basal labyrinth, suggesting a role in absorption. The Golgi apparatus is involved in the production of two main components of calcium spherules; the fibrillar material and mineralized granules. Golgi complex, rough endoplasmic reticulum (RER), ribosomes, and altered mitochondria are involved in the formation of calcium spherules. Secretory activity is indicated by the formation of dense granules containing iron and calcium salts. Lipofuscin pigment has been found in large concretions which may arise from cytoplasmic areas surrounded by endoplasmic reticulum, RER and Golgi tubules. There are three stages of excretory cells, called early, mature, and post-excretory cells. This study traces the development of granulofibrillar vacuoles up to the formation of the lipofuscin concretions and shows that excretory cells are in fact degenerating calcium cells. The fine structure of thin cells suggests that they are young calcium cells.

Topographical localization of lipofuscin pigment in the brain of the aged fat‐tailed dwarf lemur Cheirogaleus Medius and grey lesser mouse lemur Microcebus Murinus: Comparison to iron localization

Topographical localization of lipofuscin pigment in the brain of the aged fat‐tailed dwarf lemur Cheirogaleus Medius and grey lesser mouse lemur Microcebus Murinus: Comparison to iron localization

Topographical localization of lipofuscin pigment in the brain of the aged fat-tailed dwarf lemur (Cheirogaleus medius) and grey lesser mouse lemur (Microcebus murinus): comparison to iron localization.

The present study was undertaken to explore the distribution of lipofuscin in the brain of cheirogaleids by autofluorescence and compare it to other studies of iron distribution. Aged dwarf (Cheirogaleus medius) and mouse (Microcebus murinus) lemurs provide a reliable model for the study of normal and pathological cerebral aging. Accumulation of lipofuscin, an age pigment derived by lipid peroxidation, constitutes the most reliable cytological change correlated with neuronal aging. Brain sections of four aged (8-15 year old) and 3 young (2-3 year old) animals were examined. Lipofuscin accumulation was observed in the aged animals but not in the young ones. Affected regions include the hippocampus (granular and pyramidal cells), where no iron accumulation was observed, the olfactory nucleus and the olfactory bulb (mitral cells), the basal forebrain, the hypothalamus, the cerebellum (Purkinje cells), the neocortex (essentially in the pyramidal cells), and the brainstem. Even though iron is known to catalyse lipid oxidation, our data indicate that iron deposits and lipofuscin accumulation are not coincident. Different biochemical and morphological cellular compartments might be involved in iron and lipofuscin deposition. The nonuniform distribution of lipofuscin indicates that brain structures are not equally sensitive to the factors causing lipofuscin accumulation. The small size, the rapid maturity, and the relatively short life expectancy of the cheirogaleids make them a good model system in which to investigate the mechanisms of lipofuscinogenesis in primates.

The Photoreactivity of the Retinal Age Pigment Lipofuscin

The presence of the age pigment lipofuscin is associated with numerous age-related diseases. In the retina lipofuscin is located within the pigment epithelium where it is exposed to high oxygen and visible light, a prime environment for the generation of reactive oxygen species. Although we, and others, have demonstrated that retinal lipofuscin is a photoinducible generator of reactive oxygen species it is unclear how this may translate into cell damage. The position of lipofuscin within the lysosome infers that irradiated lipofuscin is liable to cause oxidative damage to either the lysosomal membrane or the lysosomal enzymes. We have found that illumination of lipofuscin with visible light is capable of extragranular lipid peroxidation, enzyme inactivation, and protein oxidation. These effects, which were pH-dependent, were significantly reduced by the addition of the antioxidants, superoxide dismutase and 1,4-diazabicyclo(2,2,2)-octane, confirming a role for both the superoxide anion and singlet oxygen. We postulate that lipofuscin may compromise retinal cell function by causing loss of lysosomal integrity and that this may be a major contributory factor to the pathology associated with retinal light damage and diseases such as age-related macular degeneration.

Independent Synthesis, Solution Behavior, and Studies on the Mechanism of Formation of a Primary Amine-Derived Fluorophore Representing Cross-linking of Proteins by (E)-4-Hydroxy-2-nonenal

Lipid peroxidation in aging and degenerative disease results in the production of 4-hydroxy-2-alkenals that modify proteins and give rise to both protein cross-linking and fluorophore generation. Recent model studies demonstrated that the major ex/em 360/430 fluorophore formed from (E)-4-hydroxy-2-nonenal (HNE) or (E)-4-hydroxy-2-hexenal (HHE) and protein lysine-based amine is a 2-alkyl-2-hydroxy-1,2-dihydropyrrol-3-one iminium 1:2 cross-link (1), a structure that is further confirmed here using 15N-labeling, and which has pH stability characteristics the same as those of lipofuscin pigments isolated from human tissues. Fluorophore generation represents an overall four-electron oxidation, requires dioxygen, and is enhanced by the presence of Cu(II). The HNE−propylamine-derived fluorophore 1a was independently synthesized from either 3,4-dioxononanal (8) or (E)-4-oxo-2-nonenal (13), providing further evidence for its assigned structure and clues to how it forms from HNE. Mechanistic studies on HNE-derived ...

Tracheobronchial Melanosis

The term “melanosis” originates from the Greek words: “melas” meaning “black,” and “osis” meaning “condition.” In medical literature, the term merely describes a condition in which there is an abnormal grayish-black or brownish-black pigmentation of an organ or a part. It does not imply that the pigment is “melanin,” although this may be true in most of the cases. Lipofuscin or other black pigment may be the cause of pigmentation. When the pigment is lipofuscin, sometimes the term “lipofuscinosis” is used; however, melanosis is the preferred term to avoid confusion caused by multiple terms. We define “tracheobronchial melanosis” (TBM) as “grayish or brownish-black pigmentation of the tracheobronchial mucosa” and describe a case of TBM with relevant review of the literature.

Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.

The fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 1-10 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.

Immunohistochemical detection of dityrosine in lipofuscin pigments in the aged human brain

Lipofuscin is a yellowish brown fluorescent pigment which is sequestered within cytoplasmic granules during aging. To examine the contribution of protein oxidation to lipofuscin accumulation, we performed immunohistochemical detection of dityrosine, which is considered one of the specific markers for protein oxidation, in lipofuscin in the aged human brain using an antibody specific to dityrosine. By characterization using competitive enzyme-linked immunosorbent assay, the specificity of the antibody to dityrosine was confirmed. None of the other tyrosine-related compounds such as l-tyrosine, 3-nitrotyrosine, 3-chlorotyrosine, or 3,4-dihydroxyphenylalanine cross-reacted with the antibody. The anti-dityrosine antibody reacted with lipofuscin granules in the pyramidal neurons of the aged human brain. The results suggest that protein oxidation by free radicals and/or peroxidases may play an important role in lipofuscin accumulation.

Estudo morfométrico do miocárdio em adultos com subnutrição protéico-energética

OBJETIVO:Comparar o tamanho das células musculares e a presença de lipofuscina no coração de adultos necropsiados, subnutridos e controles. MÉTODOS:De uma casuística inicial de 315 necropsias de adultos, foram excluídos casos com sinais de retenção hídrica, cardiopatias, hepatopatias, hipertensão e nefropatias. Subnutrição foi caracterizada pelo índice de massa corporal (IMC) <17kg/m². Fragmentos de miocárdio foram processados para morfometria computadorizada, determinando-se a espessura dos miocardiócitos e a presença de lipofuscina. RESULTADOS:Subnutridos (n=8) e controles (n=4) foram estatisticamente diferentes, respectivamente, quanto ao IMC (14,86±1,13 vs 22,02±0,9kg/m²), relação peso cardíaco/peso corporal (0,68±0,09 vs 0,54±0,07%), espessura de miocardiócitos (10,91±0,77 vs 12,90±1,82µm) e presença de lipofuscina no miocárdio (39,1 vs 54,4%). CONCLUSÃO: Comparados aos controles, adultos subnutridos têm menores espessuras de miocardiócitos e presença de lipofuscina, fenômenos que refletiriam um distúrbio metabólico com repercussões clínicas danosas em pacientes terminais.

Histologic variations in the epididymis: findings in 167 orchiectomy specimens.

Nonpathologic morphologic variations in the epididymal histology in 167 orchiectomy specimens were analyzed to assess and document the nature, frequency, and possible relation to patient age and underlying testicular pathology. Variations in histology included intranuclear eosinophilic inclusions, lipofuscin pigment, cribriform hyperplasia, Paneth cell-like metaplasia, and nuclear atypia. Intranuclear eosinophilic inclusions were observed in 72.5% of patients, and they appeared to occur at an older age than cribriform hyperplasia and Paneth cell-like metaplasia. Lipofuscin pigment was found in 32.9% of patients; this change was observed predominantly in ductuli efferentes and was more commonly associated with obstructive changes. Cribriform hyperplasia was seen in 41.9% of patients, and it occurred in 1 normal testis and in 33 testes with diverse pathologic alterations. Paneth cell-like metaplasia characterized by bright eosinophilic intracytoplasmic hyaline-like granules and globules, was present in 8.3% of patients and was accompanied by changes of obstruction in almost all instances. The globules were strongly periodic acid-Schiff positive, both before and after diastase digestion, and were negative for chromogranin A, KP-1, and MAC387 immunostains. Nuclear atypia, similar to that seen in seminal vesicles, was focally present in 13.8% of patients and tended to occur at an older age. The authors conclude that variations in epididymal morphology are fairly common and, therefore, surgical pathologists should be aware of these changes. Although exuberant in some patients, in no cases did these variations cause serious diagnostic problems.

Complex regional pain syndrome type I (RSD): pathology of skeletal muscle and peripheral nerve.

Reflex sympathetic dystrophy (RSD) (recently reclassified as complex regional pain syndrome type I) is a syndrome occurring in extremities and, when chronic, results in severe disability and untractable pain. RSD may be accompanied by neurologic symptoms even when there is no previous neurologic lesion. There is no consensus as to the pathogenic mechanism involved in RSD. To gain insight into the pathophysiology of RSD, we studied histopathology of skeletal muscle and peripheral nerve from patients with chronic RSD in a lower extremity. In eight patients with chronic RSD, an above-the-knee amputation was performed because of a nonfunctional limb. Specimens of sural nerves, tibial nerves, common peroneal nerves, gastrocnemius muscles, and soleus muscles were obtained from the amputated legs and analyzed by light and electron microscopy. In all patients, the affected leg showed similar neurologic symptoms such as spontaneous pain, hyperpathy, allodynia, paresis, and anesthesia dolorosa. The nerves showed no consistent abnormalities of myelinated fibers. In four patients, the C-fibers showed electron microscopic pathology. In all patients, the gastrocnemius and soleus muscle specimens showed a decrease of type I fibers, an increase of lipofuscin pigment, atrophic fibers, and severely thickened basal membrane layers of the capillaries. In chronic RSD, efferent nerve fibers were histologically unaffected; from afferent fibers, only C-fibers showed histopathologic abnormalities. Skeletal muscle showed a variety of histopathologic findings, which are similar to the histologic abnormalities found in muscles of patients with diabetes.

Estimating age and growth in long‐lived temperate freshwater crayfish using lipofuscin

1. In ecological studies on freshwater crayfish, determination of basic population parameters is often complicated by the lack of a suitable age estimation method.2. Previously, lipofuscin age pigment in the olfactory lobe cell masses (OLCM) of short‐lived tropical crayfish has been used for accurate age determination. Here we present the first test of this method on a longer‐lived, temperate species, the signal crayfish, Pacifastacus leniusculus.3. Confocal fluorescence microscopy and image analysis of histological sections were used to quantify OLCM lipofuscin in a reference sample of Swedish P. leniusculus from several known year‐classes, reared under naturally variable temperature conditions. Lipofuscin concentration was linearly associated with age (r2 = 92.4%) and produced much more accurate age estimates than conventional body size‐based procedures.4. A model derived from the crayfish of known‐age was used to estimate the ages of wild P. leniusculus from an English stream. The relationship between lipofuscin‐estimated age and carapace length suggested relatively slow growth in this wild population, consistent with a high population density and severe competition. The analysis also extended the known longevity of P. leniusculus to approximately 16 years.5. The lipofuscin method for determining age and growth may be widely applicable to freshwater crayfish, with probable further potential both within and outside the Crustacea.

Use of lipofuscin for resolving cohorts of western rock lobster (Panulirus cygnus)

We evaluated lipofuscin age pigment as an approach to age determination of western rock lobster (Panulirus cygnus). Lipofuscin was measured using fluorescence image analysis of olfactory lobe sections from 184 juvenile lobsters from coastal nursery reefs at Seven Mile Beach, Western Australia. Modal analysis of a lipofuscin concentration-frequency histogram for this sample suggested the presence of additional age-classes, not apparent in conventional size-frequency distributions but expected to be present from earlier tag-recapture studies. Modal progression suggested a remarkably constant average lipofuscin accumulation rate of 0.31% by volume per year in the wild lobsters, which was supported by blind-trial measurements of lipofuscin in known-age laboratory-reared specimens. Lipofuscin-based age estimates, indicating that most juvenile lobsters are 3-5 years old immediately prior to recruitment to the fishery at 76 mm carapace length, agree with the long-established relationship between puerulus settlement and lobster catch 3-4 years later. Results indicate that the lipofuscin ageing technique will permit independent assessment of current population parameter estimates and be useful for determining sexual and regional differences in these for the western rock lobster.

Selenite and selenium yeast as feed supplements to growing fattening pigs.

Twenty-four cross-bred fattening pigs weighing, on average, 24 kg were divided into three equal groups and fed a basic diet containing 0.1 mg selenium/kg. The diet of group A was supplemented with 0.3 mg selenium/kg derived from selenium yeast, group B with 0.1 mg selenium/kg from the same source, and group C with 0.3 mg/kg from sodium selenite. The pigs were slaughtered after 103 days. There was no significant difference between the mean whole blood selenium concentration of the pigs supplemented with selenium yeast neither after five nor after nine weeks of supplementation (group A, 201 and 213 micrograms/litre; group B 192 and 201 micrograms/litre, respectively), but the group supplemented with selenite had significantly lower concentrations both after five and nine weeks (158 and 159 micrograms/litre, respectively). There were no significant differences between the mean activity of glutathione peroxidase (GSH-Px; in the whole blood of the three groups (group A, 348 and 327 mukat*/litre, group B, 342 and 302 mukat/litre; group C, 332 and 300 mukat/litre, after five and nine weeks, respectively). The concentration of selenium in the liver of the pigs supplemented with organic selenium was significantly higher than in the pigs supplemented with selenite (group A, 0.54 mg/kg wet weight; group B, 0.54 mg/kg; group C, 0.40 mg/kg). No deposits of lipofuscin or ceroid pigments were observed in the histological preparations of liver and heart from any of the pigs.

Lipofuscin accumulation in cultured retinal pigment epithelial cells reduces their phagocytic capacity

Purpose. Retinal pigment epithelial (RPE) cells slowly accumulate lipofuscin pigment within their acidic vacuolar apparatus as a result of extra- and/or intralysosomal oxidative alterations of phagocytosed photoreceptor outer segments (POS) with consequent imperfect degradation of these structures. In old age, lipofuscin accumulation may become quite substantial. It has been suggested that pronounced accumulation of lipofuscin is related to decreased RPE function and, possibly, to age-related macular degeneration. The aim of the present investigation was to study whether heavy loading with lipofuscin of RPE acidic lysosomes would affect the further phagocytic ability of the cells.Methods. In the first section of the investigation, cultures of rabbit RPE cells were exposed daily to bovine UV-irradiated POS (artificial lipofuscin) for 4 weeks, resulting in a pronounced lipofuscin accumulation of the cells. Fluorescent latex beads (labelled with a red fluorophore) were added to unloaded control cultures at 0 and 4 weeks after their establishment, and to lipofuscin loaded cells after 4 weeks of feeding with artificial lipofuscin. Cellular amounts of lipofuscin, and their phagocytotic activity, were quantified by static fluorometry measuring lipofuscin-specific and red bead-specific fluorescence, respectively. The intracellular location of the beads was verified by confocal laser scanning microscopy.Results. Unloaded, and thus almost lipofuscin-free, control cells exposed to latex beads showed numerous cytoplasmic particles emitting reddish fluorescence, while few particles were taken up by cells initially loaded with artificial, POS-derived, lipofuscin. Measurement of the latex bead-specific fluorescence showed significantly (p < 0.001) higher levels in unloaded control cells than in lipofuscin-loaded ones.In the second part of the investigation, unloaded control cultures and lipofuscin-loaded cultures were exposed to native bovine Texas Red-X-labelled POS 4 weeks after the establishment of the cultures. Unloaded control cells showed a large number of cytopiasmic POS emitting reddish fluorescence, while fewer POS were phagocytosed by cells loaded with artificial lipofuscin. Measurement of the Texas Red-X-specific fluorescence, thus quantifying the phagocytic ability of the cells, showed significantly (p < 0.001) higher levels in control cells than in lipofuscin-loaded ones.Conclusions. Severe lipofuscin accumulation of RPE cells appears to result in a greatly decreased phagocytic capacity. The resulting reduction in ability to cope with the needs of the overlying photoreceptor cells, in order to eliminate the obsolete tips of their POS, may well be of significance in the development of age-related macular degeneration. Curr. Eye Res. 17:851–857, 1998.

Aging-associated Changes in Human Brain

A wide variety of anatomic and histological alterations are common in brains of aged individuals. However, identification of intrinsic aging changes--as distinct from changes resulting from cumulative environmental insult--is problematic. Some degree of neuronal and volume loss would appear to be inevitable, but recent studies have suggested that the magnitudes of such changes are much less than previously thought, and studies of dendritic complexity in cognitively intact individuals suggest continuing neuronal plasticity into the eighth decade. A number of vascular changes become more frequent with age, many attributable to systemic conditions such as hypertension and atherosclerosis. Age-associated vascular changes not clearly linked to such conditions include hyaline arteriosclerotic changes with formation of arterial tortuosities in small intracranial vessels and the radiographic changes in deep cerebral white matter known as "leukoaraiosis." Aging is accompanied by increases in glial cell activation, in oxidative damage to proteins and lipids, in irreversible protein glycation, and in damage to DNA, and such changes may underlie in part the age-associated increasing incidence of "degenerative" conditions such as Alzheimer disease and Parkinson disease. A small number of histological changes appear to be universal in aged human brains. These include increasing numbers of corpora amylacea within astrocytic processes near blood-brain or cerebrospinal fluid-brain interfaces, accumulation of the "aging" pigment lipofuscin in all brain regions, and appearance of Alzheimer-type neurofibrillary tangles (but not necessarily amyloid plaques) in mesial temporal structures.

Immunohistochemical Localization of Advanced Glycation End Products, Pentosidine, and Carboxymethyllysine in Lipofuscin Pigments of Alzheimer's Disease and Aged Neurons

Lipofuscins are intracellular fluorescent pigments accumulating in the central nervous system (CNS) with aging and degenerative processes such as Alzheimer's disease (AD). Although they are thought to be lipid peroxidation products derived from malondialdehyde, their biogenesis remains controversial. We further characterize the chemical nature of lipofuscins in brain tissues from AD patients and normal aged subjects. Advanced glycation end products (AGEs), pentosidine and carboxymethyllysine (CML), were identified by appropriate specific antibodies. They have physicochemical properties similar to those of lipofuscin and also increase with aging. Pentosidine and CML were identified in the neuronal perikarya and the extraneuroperikaryal deposits of both the AD and aged brain. Pentosidine, but not CML, was present in the fiber-like structure within the neuropil and the core of classical senile plaque. In the brain of young subjects without CNS disease, pentosidine and CML staining was faint. Pentosidine and CML co-localized with lipofuscin pigments in the neuronal perikarya of both the AD and aged brain. We demonstrate for the first time that lipofuscin is constituted not only of lipid peroxidation products but also from glycation products which may be the origin of fluorescent pigments. Lipofuscins should thus be considered as fluorescent pigments generated by lipid- and sugar-derived Schiff base-protein polymers.

Chronic Cerebral Hypoperfusion: Pathological and Behavioral Consequences

OBJECTIVE: Although the effects of acute ischemic insults to the brain are well known, the effects related to chronic ischemia are poorly delineated. The pathological and behavioral changes induced by a chronic noninfarctional reduction in cerebral blood flow of 25 to 50% maintained for 6 months were assessed. METHODS: In each of 18 male Sprague-Dawley rats, an arteriovenous fistula was created in the neck via an anastomosis between the right external jugular vein and the right common carotid artery to induce cerebral hypoperfusion. Nineteen age-matched animals comprised a control group. Six months after surgery, the animals were examined using light and electron microscopic techniques, as well as via a battery of behavioral tests (motor, open field, and T-maze). RESULTS: Examination of the hippocampus by using light microscopy revealed disorganization of the CA1 sector with an increased number of astrocytes. Transmission electron microscopy of the CA1 region demonstrated neurons with increased lipofuscin pigment and central nucleoli and astrocytes with more numerous cytosolic mitochondria. Motor performance testing revealed no gross motor deficits, although open-field assessment demonstrated increased exploratory behavior in rats with fistulas. Finally, T-maze testing results suggested that errors in working memory were more common in rats undergoing chronic cerebral hypoperfusion (P < 0.05). CONCLUSIONS: These findings suggest that chronic reductions in cerebral blood flow of a magnitude previously thought to be harmless to neurons (i.e., reduced by 25-50%) do alter neuronal structure and affect whole animal behavior. Such a scenario may be responsible for a symptomatology secondary to arteriovenous steal and severe carotid stenoses. The mechanisms are still unknown.

Chronic cerebral hypoperfusion: pathological and behavioral consequences.

Although the effects of acute ischemic insults to the brain are well known, the effects related to chronic ischemia are poorly delineated. The pathological and behavioral changes induced by a chronic noninfarctional reduction in cerebral blood flow of 25 to 50% maintained for 6 months were assessed. In each of 18 male Sprague-Dawley rats, an arteriovenous fistula was created in the neck via an anastomosis between the right external jugular vein and the right common carotid artery to induce cerebral hypoperfusion. Nineteen age-matched animals comprised a control group. Six months after surgery, the animals were examined using light and electron microscopic techniques, as well as via a battery of behavioral tests (motor, open field, and T-maze). Examination of the hippocampus by using light microscopy revealed disorganization of the CA1 sector with an increased number of astrocytes. Transmission electron microscopy of the CA1 region demonstrated neurons with increased lipofuscin pigment and central nucleoli and astrocytes with more numerous cytosolic mitochondria. Motor performance testing revealed no gross motor deficits, although open-field assessment demonstrated increased exploratory behavior in rats with fistulas. Finally, T-maze testing results suggested that errors in working memory were more common in rats undergoing chronic cerebral hypoperfusion (P < 0.05). These findings suggest that chronic reductions in cerebral blood flow of a magnitude previously thought to be harmless to neurons (i.e., reduced by 25-50%) do alter neuronal structure and affect whole animal behavior. Such a scenario may be responsible for a symptomatology secondary to arteriovenous steal and severe carotid stenoses. The mechanisms are still unknown.