Altered Blood Flow in the Ophthalmic and Internal Carotid Arteries in Patients with Age-Related Macular Degeneration Measured Using Noncontrast MR Angiography at 7T.

BACKGROUND AND PURPOSE: Age-related macular degeneration is associated with reduced perfusion of the eye; however, the role of altered blood flow in the upstream ophthalmic or internal carotid arteries is unclear. We used ultra-high-field MR imaging to investigate whether the diameter of and blood flow in the ophthalmic artery and/or the ICA are altered in age-related macular degeneration and whether any blood flow changes are associated with disease progression. MATERIALS AND METHODS: Twenty-four patients with age-related macular degeneration and 13 similarly-aged healthy controls participated. TOF and high-resolution dynamic 2D phase-contrast MRA (0.26 × 0.26 × 2mm3, 100-ms effective sampling rate) was acquired at 7T. Vessel diameters were calculated from cross-sectional areas in phase-contrast acquisitions. Blood flow time-series were measured across the cardiac cycle. RESULTS: The ophthalmic artery vessel diameter was found to be significantly smaller in patients with age-related macular degeneration than in controls. Volumetric flow through the ophthalmic artery was significantly lower in patients with late age-related macular degeneration, with a significant trend of decreasing volumetric ophthalmic artery flow rates with increasing disease severity. The resistance index was significantly greater in patients with age-related macular degeneration than in controls in the ophthalmic artery. Flow velocity through the ophthalmic artery and ICA was significantly higher in patients with age-related macular degeneration. Ophthalmic artery blood flow as a percentage of ipsilateral ICA blood flow was nearly double in controls than in patients with age-related macular degeneration. CONCLUSIONS: These findings support the hypothesis that vascular changes upstream to the eye are associated with the severity of age-related macular degeneration. Additional investigation into the potential causality of this relationship and whether treatments that improve ocular circulation slow disease progression is warranted.

Triglyceride-rich lipoproteins prevent septic death in rats.

Triglyceride-rich lipoproteins bind and inactive bacterial endotoxin in vitro and prevent death when given before a lethal dose of endotoxin in animals. However, lipoproteins have not yet been demonstrated to improve survival in polymicrobial gram-negative sepsis. We therefore tested the ability of triglyceride-rich lipoproteins to prevent death after cecal ligation and puncture (CLP) in rats. Animals were given bolus infusions of either chylomicrons (1 g triglyceride/kg per 4 h) or an equal volume of saline for 28 h after CLP. Chylomicron infusions significantly improved survival (measured at 96 h) compared with saline controls (80 vs 27%, P < or = 0.03). Chylomicron infusions also reduced serum levels of endotoxin, measured 90 min (26 +/- 3 vs 136 +/- 51 pg/ml, mean +/- SEM, P < or = 0.03) and 6 h (121 +/- 54 vs 1,026 +/- 459 pg/ml, P < or = 0.05) after CLP. The reduction in serum endotoxin correlated with a reduction in serum tumor necrosis factor, measured 6 h after CLP (0 +/- 0 vs 58 +/- 24 pg/ml, P < or = 0.03), suggesting that chylomicrons improve survival in this model by limiting macrophage exposure to endotoxin and thereby reducing secretion of inflammatory cytokines. Infusions of a synthetic triglyceride-rich lipid emulsion (Intralipid; KabiVitrum, Inc., Alameda, CA) (1 g triglyceride/kg) also significantly improved survival compared with saline controls (71 vs 27%, P < or = 0.03). These data demonstrate that triglyceride-rich lipoproteins can protect animals from lethal polymicrobial gram-negative sepsis.

The cytochrome oxidase superfamily of redox-driven proton pumps.

Most respiratory oxidases of eukaryotic and prokaryotic organisms are members of a superfamily of enzymes that couple the redox energy available from the reduction of molecular oxygen to the mechanism of pumping protons across the membrane. The recent applications of site-directed mutagenesis and of a variety of spectroscopic techniques have allowed major advances in our understanding of the structure and function of these proteins.

Relative toxicity of gossypol enantiomers in laying and broiler breeder hens.

Gossypol, a natural component of cottonseed meal, exists in positive (+) or negative (-) enantiomeric forms, and their levels and ratio could be altered by developing new genetic strains of cotton. Two experiments were conducted to determine the relative toxicity of the individual gossypol enantiomers in laying and broiler breeder hens. In the first experiment, 25 individually caged Hy-Line W-36 forty-three-week-old laying hens were fed a standard corn-soy diet supplemented with either no gossypol or the individual enantiomers at 200 and 400 mg/kg of diet for 20 d (5 hens/treatment). In the second experiment, 15 individually caged Cobb 500 fast-feathering 44-wk-old broiler breeder hens were fed a standard corn-soy-wheat middlings diet supplemented with either no gossypol or the individual enantiomers at 400 mg/kg of diet for 18 d (5 hens/treatment). In both experiments, feed intake, egg production, and egg weight were determined daily. All eggs were individually opened and scored for yolk discoloration. At the end of both experiments, several organ and tissue samples were collected for gossypol analyses. In both experiments, the addition of (+)-gossypol to the diet reduced egg production. Only laying and broiler breeder hens fed (+)-gossypol produced eggs with severe yolk discoloration (score > or = 4). Total feed intake was lower (P < 0.05) in laying hens fed the 400 mg/kg level of (+)-gossypol compared with laying hens fed the other dietary treatments. In contrast, broiler breeder hens consumed less of the diet supplemented with (-)-gossypol. In both experiments, tissue accumulation of (+)-gossypol was higher than (-)-gossypol, with the exception of bile and excreta. The results suggest that in hens the ingestion of (+)-gossypol has a greater effect on egg yolk discoloration than the consumption of (-)-gossypol.

Finite element modeling, validation, and parametric investigations of a retinal reattachment stent.

A new retinal reattachment surgical procedure is based on a stent that is deployed to press the retina back in place. An eye-stent finite element model studied the strain induced by the stent on retina. Finite element model simulations were performed for several stent geometric configurations (number of loops, wire diameter, and intraocular pressure). The finite element model was validated against experiment. Parametric studies demonstrated that stents could be successfully designed so that the maximum strain would be below permanent damage strain threshold of 2%.

Acoustical Method of Whole-Body Hydration Status Monitoring.

An acoustical handheld hydration monitor (HM) for assessing the water balance of the human body was developed. Dehydration is a critical public health problem. Many elderly over age of 65 are particularly vulnerable as are infants and young children. Given that dehydration is both preventable and reversible, the need for an easy-to-perform method for the detection of water imbalance is of the utmost clinical importance. The HM is based on an experimental fact that ultrasound velocity in muscle is a linear function of water content and can be referenced to the hydration status of the body. Studies on the validity of HM for the assessment of whole-body hydration status were conducted in the Appalachian State University, USA, on healthy young adults and on elderly subjects residing at an assisted living facility. The HM was able to track changes in total body water during periods of acute dehydration and rehydration in athletes and day-to-day and diurnal variability of hydration in elderly. Results of human studies indicate that HM has a potential to become an efficient tool for detecting abnormal changes in the body hydration status.

Cerebral amyloid induces aberrant axonal sprouting and ectopic terminal formation in amyloid precursor protein transgenic mice.

A characteristic feature of Alzheimer's disease (AD) is the formation of amyloid plaques in the brain. Although this hallmark pathology has been well described, the biological effects of plaques are poorly understood. To study the effect of amyloid plaques on axons and neuronal connectivity, we have examined the axonal projections from the entorhinal cortex in aged amyloid precursor protein (APP) transgenic mice that exhibit cerebral amyloid deposition in plaques and vessels (APP23 mice). Here we report that entorhinal axons form dystrophic boutons around amyloid plaques in the entorhinal termination zone of the hippocampus. More importantly, entorhinal boutons were found associated with amyloid in ectopic locations within the hippocampus, the thalamus, white matter tracts, as well as surrounding vascular amyloid. Many of these ectopic entorhinal boutons were immunopositive for the growth-associated protein GAP-43 and showed light and electron microscopic characteristics of axonal terminals. Our findings suggest that (1) cerebral amyloid deposition has neurotropic effects and is the main cause of aberrant sprouting in AD brain; (2) the magnitude and significance of sprouting in AD have been underestimated; and (3) cerebral amyloid leads to the disruption of neuronal connectivity which, in turn, may significantly contribute to AD dementia.

Spontaneous hemorrhagic stroke in a mouse model of cerebral amyloid angiopathy.

A high risk factor for spontaneous and often fatal lobar hemorrhage is cerebral amyloid angiopathy (CAA). We now report that CAA in an amyloid precursor protein transgenic mouse model (APP23 mice) leads to a loss of vascular smooth muscle cells, aneurysmal vasodilatation, and in rare cases, vessel obliteration and severe vasculitis. This weakening of the vessel wall is followed by rupture and bleedings that range from multiple, recurrent microhemorrhages to large hematomas. Our results demonstrate that, in APP transgenic mice, the extracellular deposition of neuron-derived beta-amyloid in the vessel wall is the cause of vessel wall disruption, which eventually leads to parenchymal hemorrhage. This first mouse model of CAA-associated hemorrhagic stroke will now allow development of diagnostic and therapeutic strategies.

Strong-field and integral spin-ligand complexes of the cytochrome bo quinol oxidase in Escherichia coli membrane preparations.

The cytochrome bo-type terminal oxidase of Escherichia coli is an analogue of mammalian aa3-type cytochrome c oxidase. The catalytic core of both enzymes is a binuclear site containing a penta-coordinate heme (heme o or a3) and copper (CuB). Herein we report on UV-visible and magnetic properties of ligand complexes of the binuclear site of cytochrome bo. Cyanide, sulfide, and azide react with the Fe(3+)-Cu+ center to give EPR-detectable low-spin complexes, analogous to those formed by cytochrome aa3. Analyses of the ligand fields of these complexes indicate that heme o has a single axial histidine ligand. Cyanide and azide react with the Fe(3+)-Cu2+ center to yield forms observable via UV-visible spectroscopy but not EPR. With formate and fluoride, cytochrome bo forms integral spin complexes similar to those of cytochrome aa3. These complexes have UV-visible characteristics of high-spin species, but EPR spectra show features which appear to correspond to transitions within an integral spin multiplet. Cytochrome bo forms another integral spin complex with azide and NO which is nearly identical to the azide-NO species in cytochrome aa3. This suggests that the binuclear centers of the two enzymes are quite similar.

Relative toxicity of gossypol enantiomers in broilers.

Use of cottonseed meal in poultry diets has been avoided in large part because of fear of gossypol toxicity. Gossypol exists naturally as a mixture of 2 enantiomers that exhibit different biological activities. Two experiments were conducted to determine the relative toxicity of gossypol enantiomers on broilers. In the first experiment, 3-d-old broilers were fed a standard diet containing 0, 100, 200, 300, or 400 mg of gossypol from gossypol acetic acid per kilogram of diet from 3 to 42 d of age. This form of gossypol contains both enantiomers in an equimolar ratio. Each dietary treatment consisted of 6 replicate pens of 4 birds. In the second experiment, 3-d-old broilers were divided into 15 pens of 4 birds each and fed a standard diet supplemented with either no gossypol or one of the gossypol enantiomers at 200 or 400 mg/kg of diet from 3 to 21 d of age. In both experiments, feed intake and BW gain were measured. In addition, several organ and tissue samples were collected at 21 d (experiments 1 and 2) and 42 d (experiment 1) of age and analyzed for gossypol. In experiment 1, feed consumption and BW gain were reduced (P < 0.05) at 21 and 42 d for the birds fed the highest level of gossypol. The concentration of gossypol in the heart, kidney, and plasma were equivalent at 21 and 42 d of age. In experiment 2, total feed consumption was reduced only in birds consuming (-)-gossypol, but BW gains were lower for birds fed either enantiomer. However, (-)-gossypol was more detrimental to growth than (+)-gossypol. The liver had the highest tissue concentration of both enantiomers, and accumulation of (+)-gossypol was higher than (-)-gossypol in all tissues examined. No racemization of the enantiomers was apparent in the tissues analyzed. Our results indicated that both gossypol enantiomers were toxic to broilers but that (-)-gossypol was more harmful to efficient broiler production than (+)-gossypol.

Cognitive decline strongly correlates with cortical atrophy in Alzheimer's dementia.

Alzheimer's disease (AD) is characterized by progressive dementia and distinct neuropathology at autopsy. In order to test the relationship between dementia severity and loss of brain volumes, we prospectively documented the neurological/medical health of 26 male and 26 female controls and AD cases, and evaluated a subset of controls and AD cases using the Mini Mental State Examination (MMSE). At autopsy, Consortium to Establish a Registry for Alzheimer's Disease (CERAD) criteria confirmed diagnoses in 33 AD cases and 19 controls, and using unbiased stereology we quantified total volumes of cortical gray matter, subcortical grey matter including white matter, and forebrain. For ages of death between 50 to 100 years, controls showed minor cortical atrophy in the absence of cognitive decline. Cortical atrophy in AD cases was 20 to 25% greater than that in controls; AD patients dying at older ages showed less severe cortical atrophy than those dying at younger ages. Across all AD cases there was a strong correlation between cognitive performance on the Mini Mental State Examination and cortical volume loss. These findings confirm fundamental differences in the temporal patterns of cortical volume loss in aging and AD, and support cortical degeneration as the primary basis for cognitive decline in AD.

Hippocampal neuron and synaptophysin-positive bouton number in aging C57BL/6 mice.

A loss of hippocampal neurons and synapses had been considered a hallmark of normal aging and, furthermore, to be a substrate of age-related learning and memory deficits. Recent stereological studies in humans have shown that only a relatively minor neuron loss occurs with aging and that this loss is restricted to specific brain regions, including hippocampal subregions. Here, we investigate these age-related changes in C57BL/6J mice, one of the most commonly used laboratory mouse strains. Twenty-five mice (groups at 2, 14, and 28-31 months of age) were assessed for Morris water-maze performance, and modern stereological techniques were used to estimate total neuron and synaptophysin-positive bouton number in hippocampal subregions at the light microscopic level. Results revealed that performance in the water maze was largely maintained with aging. No age-related decline was observed in number of dentate gyrus granule cells or CA1 pyramidal cells. In addition, no age-related change in number of synaptophysin-positive boutons was observed in the molecular layer of the dentate gyrus or CA1 region of hippocampus. We observed a significant correlation between dentate gyrus synaptophysin-positive bouton number and water-maze performance. These results demonstrate that C57BL/6J mice do not exhibit major age-related deficits in spatial learning or hippocampal structure, providing a baseline for further study of mouse brain aging.

Stereological analysis of astrocyte and microglia in aging mouse hippocampus.

Recent evidence suggests neuroglia-mediated inflammatory mechanisms may stimulate neurodegenerative processes in mammalian brain during aging. To test the hypothesis that the number of microglia and astrocytes increase in the hippocampus during normal aging, unbiased stereological techniques were used to estimate total cell number in hippocampal subregions (CA1, dentate gyrus and hilus) of male C57BL/6J mice of different ages: 4-5 months, 13-14 months and 27-28 months. Immunocytochemical visualization for microglia and astrocytes were via Mac-1 and GFAP antibody, respectively. Estimates of total microglia and astrocyte number were assessed using the optical fractionator. No statistically significant age differences were found in the numbers of microglia or astrocytes in the hippocampal regions sampled. These findings suggest that age-related increases in the total numbers of hippocampal microglia and astrocytes is not causal for observed age-related increases in cytokine response.

The formate complex of the cytochrome bo quinol oxidase of Escherichia coli exhibits a 'g = 12' EPR feature analogous to that of 'slow' cytochrome oxidase.

The cytochrome bo quinol oxidase of Escherichia coli is homologous in sequence and in structure to cytochrome aa3 type cytochrome oxidase in subunit I, which contains the catalytic core. The cytochrome bo enzyme forms a formate complex which exhibits 'g = 12' and 'g = 2.9' EPR signals at X band; similar signals have previously been observed only in association with the 'slow' and formate-ligand states of cytochrome oxidase. These signals arise from transitions within integral spin multiples identified with the homologous heme-copper binuclear catalytic centers in both enzymes.

The highly conserved methionine of subunit I of the heme-copper oxidases is not at the heme-copper dinuclear center: mutagenesis of M110 in subunit I of cytochrome bo3-type ubiquinol oxidase from Escherichia coli.

A common feature within the heme-copper oxidase superfamily is the dinuclear heme-copper center. Analysis via extended X-ray absorption fine structure (EXAFS) has led to the proposal that sulfur may be bound to CuB, a component of the dinuclear center, and a highly conserved methionine (M110 in the E. coli oxidase) in subunit I has been proposed as the ligand. Recent models of subunit I, however, suggest that this residue is unlikely to be near CuB, but is predicted to be near the low spin heme component of the heme-copper oxidases. In this paper, the role of M110 is examined by spectroscopic analyses of site-directed mutants of the bo3-type oxidase from Escherichia coli. The results show that M110 is a non-essential residue and suggest that it is probably not near the heme-copper dinuclear center.

Transgenic approaches to model Alzheimer's disease.

Two transgenic mouse lines were generated which express human APP751 containing familial Alzheimer's disease (AD) mutations in brain neurons. These mice develop pathological features reminiscent of AD. The degree of pathology depends on both expression levels and specific mutations. In mice with more advanced pathology (APP 23), typical plaques appear at six months which increase with age and are Congo Red positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. A quantitative analysis of degenerative changes by state-of-the-art unbiased stereological methods revealed a significant reduction in neuronal cell bodies of the CA1 field of the hippocampus when compared to controls. This reduction is directly related to plaque load. When subjected to analysis in the Morris water maze, 18 month old APP 23 mice show a significant increase in platform finding latency throughout the entire trial when compared to non-transgenic littermates.

Activity-regulated cytoskeletal-associated protein is localized to recently activated excitatory synapses.

Activity-regulated, cytoskeletal-associated protein (Arc) is an immediate early gene induced in excitatory circuits following behavioral episodes. Arc mRNA is targeted to activated regions of the dendrite after long-term potentiation (LTP) of the dentate gyrus, a process dependent on NMDA receptor activation. We used post-embedding immunogold electron microscopy (EM) to test whether synaptic Arc expression patterns are selectively modified by plasticity. Consistent with previous light microscopic observations, Arc protein was rapidly induced in the dentate gyrus following LTP-producing stimulation of the perforant path and was detectable in granule cell nuclei, somata and dendrites after two hours of high frequency stimulation. Post-embedding EM revealed Arc immunogold labeling in three times as many spines in the middle molecular layer of the stimulated dentate gyrus than in either the ipsilateral outer molecular layer or the contralateral middle and outer molecular layers. This upregulation did not occur with low frequency stimulation of the perforant path. Therefore Arc protein localization may be a powerful tool to isolate recently activated dendritic spines.

No hippocampal neuron or synaptic bouton loss in learning-impaired aged beta-amyloid precursor protein-null mice.

Aged beta-amyloid precursor protein-null mice were used to investigate the relationship between beta-amyloid precursor protein, hippocampal neuron and synaptic bouton number, and cognitive function. Learning and memory performance of aged beta-amyloid precursor protein-null mice and age-matched controls were assessed in the Morris water maze. Beta-amyloid precursor protein-null mice demonstrated impaired task acquisition as measured by significantly longer swim path lengths, a higher percentage of failed trials, and more frequent thigmotaxis behavior than controls. In a subsequent probe trial, beta-amyloid precursor protein-null mice spent significantly less time in the old goal quadrant, and made fewer crossings over the old platform location than did controls. No differences in motor or visual skills were observed which could account for the performance differences. In light of these findings and previous evidence for a role of beta-amyloid precursor protein in neuronal maintenance and synaptogenesis, we pursued the hypothesis that the learning impairment of beta-amyloid precursor protein-null mice may be a reflection of differences in neuron or synaptophysin-positive presynaptic bouton number. Thus, unbiased stereological analysis was used to estimate neuron and synaptic bouton number in dentate gyrus and hippocampal CA1 of the behaviorally characterized mice. No difference in neuron or synaptophysin-positive presynaptic bouton number was found between the beta-amyloid precursor protein-null mice and age-matched controls. Our results suggest that the learning impairment of beta-amyloid precursor protein-null mice is not mediated by a loss of hippocampal neurons or synaptic boutons.

Structural brain aging in inbred mice: potential for genetic linkage.

To identify genetic factors involved in brain aging, we have initiated studies assessing behavioral and structural changes with aging among inbred mouse strains. Cognitive performance of C57BL/6J mice is largely maintained with aging, and stereological analysis revealed no significant age-related change in neuron number, synaptic bouton number or glial number in the hippocampus. Moreover, no change in cortical neuron number and cholinergic basal forebrain neuron number has been found in this strain. 129Sv/J mice have more pronounced age-related cognitive deficits, although hippocampal and basal cholinergic forebrain neuron number also appear unchanged with aging. Differences in neurogenesis and neuron vulnerability in the adult CNS of C57BL/6, 129/Sv and other inbred strains have been reported, which in turn may have important consequences for brain aging. Age-related lesions, such as thalamic eosinophilic inclusions and hippocampal clusters of polyglucosan bodies also vary greatly among inbred strains although the functional significance of these lesions is not clear. The continued assessment of such age-related structural and behavioral changes among inbred mouse strains offers the potential to identify genes that control age-related changes in brain structure and function.