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Objective: To evaluate whether an imaging classifier for radiology practice can improve lung nodule classification and follow-up., Methods: A machine learning classifier was developed and trained using imaging data from the National Lung Screening Trial (NSLT) to produce a malignancy risk score (malignancy Similarity Index [mSI]) for individual lung nodules. In addition to NLST cohorts, external cohorts were developed from a tertiary referral lung cancer screening program data set and an external nonscreening data set of all nodules detected on CT. Performance of the mSI combined with Lung-RADS was compared with Lung-RADS alone and the Mayo and Brock risk calculators., Results: We analyzed 963 subjects and 1,331 nodules across these cohorts. The mSI was comparable in accuracy (area under the curve = 0.89) to existing clinical risk models (area under the curve = 0.86-0.88) and independently predictive in the NLST cohort of 704 nodules. When compared with Lung-RADS, the mSI significantly increased sensitivity across all cohorts (25%-117%), with significant increases in specificity in the screening cohorts (17%-33%). When used in conjunction with Lung-RADS, use of mSI would result in earlier diagnoses and reduced follow-up across cohorts, including the potential for early diagnosis in 42% of malignant NLST nodules from prior-year CT scans., Conclusion: A computer-assisted diagnosis software improved risk classification from chest CTs of screening and incidentally detected lung nodules compared with Lung-RADS. mSI added predictive value independent of existing radiological and clinical variables. These results suggest the generalizability and potential clinical impact of a tool that is straightforward to implement in practice., (Copyright © 2022 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

Seizure-driven brain damage in epilepsy accumulates over time, especially in the hippocampus, which can lead to sclerosis, cognitive decline, and death. Excitotoxicity is the prevalent model to explain ictal neurodegeneration. Current labeling technologies cannot distinguish between excitotoxicity and hypoxia, however, because they share common molecular mechanisms. This leaves open the possibility that undetected ischemic hypoxia, due to ictal blood flow restriction, could contribute to neurodegeneration previously ascribed to excitotoxicity. We tested this possibility with Confocal Laser Endomicroscopy (CLE) and novel stereological analyses in several models of epileptic mice. We found a higher number and magnitude of NG2+ mural-cell mediated capillary constrictions in the hippocampus of epileptic mice than in that of normal mice, in addition to spatial coupling between capillary constrictions and oxidative stressed neurons and neurodegeneration. These results reveal a role for hypoxia driven by capillary blood flow restriction in ictal neurodegeneration.

Research indicates that female risk of developing Alzheimer's disease (AD) is greater than that of males. Moderate reduction of calorie intake, known as calorie restriction (CR), reduces pathology in AD mouse models and is a potentially translatable prevention measure for individuals at-risk for AD, as well as an important tool for understanding how the brain endogenously attenuates age-related pathology. Whether sex influences the response to CR remains unknown. In this study, we assessed the effect of CR on beta-amyloid peptide (Aβ) pathology and hippocampal CA1 neuron specific gene expression in the Tg2576 mouse model of cerebral amyloidosis. Relative to ad libitum (AL) feeding, CR feeding significantly reduced hippocampal Aβ burden in 15-month-old female, but not age-matched male, Tg2576 mice. Sustained CR also significantly reduced expression of presenilin enhancer 2 (Psenen) and presenilin 1, components of the γ-secretase complex, in Tg2576 females. These results indicate that long-term CR significantly reduces age-dependent female Tg2576 Aβ pathology, which is likely to involve CR-mediated reductions in γ-secretase-dependent amyloid precursor protein (APP) metabolism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Gorillas include separate eastern (Gorilla beringei) and western (Gorilla gorilla) African species that diverged from each other approximately 2 million years ago. Although anatomical, genetic, behavioral, and socioecological differences have been noted among gorilla populations, little is known about variation in their brain structure. This study examines neuroanatomical variation between gorilla species using structural neuroimaging. Postmortem magnetic resonance images were obtained of brains from 18 captive western lowland gorillas (Gorilla gorilla gorilla), 15 wild mountain gorillas (Gorilla beringei beringei), and 3 Grauer's gorillas (Gorilla beringei graueri) (both wild and captive). Stereologic methods were used to measure volumes of brain structures, including left and right frontal lobe gray and white matter, temporal lobe gray and white matter, parietal and occipital lobes gray and white matter, insular gray matter, hippocampus, striatum, thalamus, each hemisphere and the vermis of the cerebellum, and the external and extreme capsules together with the claustrum. Among the species differences, the volumes of the hippocampus and cerebellum were significantly larger in G. gorilla than G. beringei. These anatomical differences may relate to divergent ecological adaptations of the two species. Specifically, G. gorilla engages in more arboreal locomotion and thus may rely more on cerebellar circuits. In addition, they tend to eat more fruit and have larger home ranges and consequently might depend more on spatial mapping functions of the hippocampus., (© 2015 Wiley Periodicals, Inc.)

Motor learning requires protein synthesis within the primary motor cortex (M1). Here, we show that the immediate early gene Arc/Arg3.1 is specifically induced in M1 by learning a motor skill. Arc mRNA was quantified using a fluorescent in situ hybridization assay in adult Long-Evans rats learning a skilled reaching task (SRT), in rats performing reaching-like forelimb movement without learning (ACT) and in rats that were trained in the operant but not the motor elements of the task (controls). Apart from M1, Arc expression was assessed within the rostral motor area (RMA), primary somatosensory cortex (S1), striatum (ST) and cerebellum. In SRT animals, Arc mRNA levels in M1 contralateral to the trained limb were 31% higher than ipsilateral (p<0.001), 31% higher than in the contralateral M1 of ACT animals (p<0.001) and 48% higher than in controls (p<0.001). Arc mRNA expression in SRT was positively correlated with learning success between two sessions (r=0.52; p=0.026). For RMA, S1, ST or cerebellum no significant differences in Arc mRNA expression were found between hemispheres or across behaviors. As Arc expression has been related to different forms of cellular plasticity, these findings suggest a link between M1 Arc expression and motor skill learning in rats., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Repetitive in vivo imaging in mice has become an indispensable tool for studying dynamic changes in structure and function of the brain. We describe a head fixation system, which allows rapid re-localization of previously imaged regions of interest (ROIs) within the brain. Such ROIs can be automatically relocated and imaged over weeks to months with negligible rotational change and only minor translational errors. Previously stored imaging positions can be fully automated re-localized within a few seconds. This automated rapid and accurate relocation simplifies image acquisition and post-processing in longitudinal imaging experiments. Moreover, as the laser is only used for data acquisition and not for finding previously imaged ROIs, the risk of laser induced tissue damage and photobleaching is greatly reduced. Thus, here described head fixation device appears well suited for in vivo repetitive long-term imaging in rodent brain., (Copyright © 2011 Elsevier B.V. All rights reserved.)

APPPS1 transgenic mice develop amyloid-β 42 (Aβ42)-driven early-onset cerebral β-amyloidosis. Stereological analysis of neocortical neuron number in groups of 2-, 10-, and 17-month-old APPPS1 mice did not reveal any changes compared with wild-type control animals despite massive amyloid-β (Aβ) load and disrupted cytoarchitecture. However, in subregions with high neuron density such as the granule cell layer of the dentate gyrus, modest but significant neuron loss was found, reminiscent of findings in previously published mouse models with late onset cerebral β-amyloidosis and predominant amyloid-β 40 (Aβ40) expression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Context: Autism often involves early brain overgrowth, including the prefrontal cortex (PFC). Although prefrontal abnormality has been theorized to underlie some autistic symptoms, the cellular defects that cause abnormal overgrowth remain unknown., Objective: To investigate whether early brain overgrowth in children with autism involves excess neuron numbers in the PFC. DESIGN, SETTING, AND CASES: Postmortem prefrontal tissue from 7 autistic and 6 control male children aged 2 to 16 years was examined by expert anatomists who were blinded to diagnostic status. Number and size of neurons were quantified using stereological methods within the dorsolateral (DL-PFC) and mesial (M-PFC) subdivisions of the PFC. Cases were from the eastern and southeastern United States and died between 2000 and 2006., Main Outcome Measures: Mean neuron number and size in the DL-PFC and M-PFC were compared between autistic and control postmortem cases. Correlations of neuron number with deviation in brain weight from normative values for age were also performed., Results: Children with autism had 67% more neurons in the PFC (mean, 1.94 billion; 95% CI, 1.57-2.31) compared with control children (1.16 billion; 95% CI, 0.90-1.42; P = .002), including 79% more in DL-PFC (1.57 billion; 95% CI, 1.20-1.94 in autism cases vs 0.88 billion; 95% CI, 0.66-1.10 in controls; P = .003) and 29% more in M-PFC (0.36 billion; 95% CI, 0.33-0.40 in autism cases vs 0.28 billion; 95% CI, 0.23-0.34 in controls; P = .009). Brain weight in the autistic cases differed from normative mean weight for age by a mean of 17.6% (95% CI, 10.2%-25.0%; P = .001), while brains in controls differed by a mean of 0.2% (95% CI, -8.7% to 9.1%; P = .96). Plots of counts by weight showed autistic children had both greater total prefrontal neuron counts and brain weight for age than control children., Conclusion: In this small preliminary study, brain overgrowth in males with autism involved an abnormal excess number of neurons in the PFC.

Extracellular deposition of the amyloid-β peptide (Aβ) in the brain parenchyma is a hallmark lesion of Alzheimer's disease (AD) and a predictive marker for the progression of preclinical to symptomatic AD. Here, we used multiphoton in vivo imaging to study Aβ plaque formation in the brains of 3- to 4-month-old APPPS1 transgenic mice over a period of 6 months. A novel head fixation system provided robust and efficient long-term tracking of single plaques over time. Results revealed an estimated rate of 35 newly formed plaques per cubic millimeter of neocortical volume per week at 4-5 months of age. At later time points (i.e., in the presence of increasing cerebral β-amyloidosis), the number of newly formed plaques decreased. On average, both newly formed and existing plaques grew at a similar growth rate of 0.3 μm (radius) per week. A solid knowledge of the dynamics of cerebral β-amyloidosis in mouse models provides a powerful tool to monitor preclinical Aβ targeting therapeutic strategies and eases the interpretation of diagnostic amyloid imaging in humans.

Alzheimer's disease is characterized by numerous pathological abnormalities, including amyloid beta (Abeta) deposition in the brain parenchyma and vasculature. In addition, intracellular Abeta accumulation may affect neuronal viability and function. In this study, we evaluated the effects of different forms of Abeta on cognitive decline by analyzing the behavioral induction of the learning-related gene Arc/Arg3.1 in three different transgenic mouse models of cerebral amyloidosis (APPPS1, APPDutch, and APP23). Following a controlled spatial exploration paradigm, reductions in both the number of Arc-activated neurons and the levels of Arc mRNA were seen in the neocortices of depositing mice from all transgenic lines (deficits ranging from 14 to 26%), indicating an impairment in neuronal encoding and network activation. Young APPDutch and APP23 mice exhibited intracellular, granular Abeta staining that was most prominent in the large pyramidal cells of cortical layer V; these animals also had reductions in levels of Arc. In the dentate gyrus, striking reductions (up to 58% in aged APPPS1 mice) in the number of Arc-activated cells were found. Single-cell analyses revealed both the proximity to fibrillar amyloid in aged mice, and the transient presence of intracellular granular Abeta in young mice, as independent factors that contribute to reduced Arc levels. These results provide evidence that two independent Abeta pathologies converge in their impact on cognitive function in Alzheimer's disease.

Age-related impairments in hippocampus-dependent learning and memory tasks are not associated with a loss of hippocampal neurons, but may be related to alterations in synaptic integrity. Here we used stereological techniques to estimate spine number in hippocampal subfields using immunostaining for the spine-associated protein, spinophilin, as a marker. Quantification of the immunoreactive profiles was performed using the optical disector/fractionator technique. Aging was associated with a modest increase in spine number in the molecular layer of the dentate gyrus and CA1 stratum lacunosum-moleculare. By comparison, spinophilin protein levels in the hippocampus, measured by Western blot analysis, failed to differ as a function of age. Neither the morphological nor the protein level data were correlated with spatial learning ability across individual aged rats. The results extend current evidence on synaptic integrity in the aged brain, indicating that a substantial loss of dendritic spines and spinophilin protein in the hippocampus are unlikely to contribute to age-related impairment in spatial learning.

Microglial cells aggregate around amyloid plaques in Alzheimer's disease, but, despite their therapeutic potential, various aspects of their reactive kinetics and role in plaque pathogenesis remain hypothetical. Through use of in vivo imaging and quantitative morphological measures in transgenic mice, we demonstrate that local resident microglia rapidly react to plaque formation by extending processes and subsequently migrating toward plaques, in which individual transformed microglia somata remain spatially stable for weeks. The number of plaque-associated microglia increased at a rate of almost three per plaque per month, independent of plaque volume. Larger plaques were surrounded by larger microglia, and a subset of plaques changed in size over time, with an increase or decrease related to the volume of associated microglia. Far from adopting a more static role, plaque-associated microglia retained rapid process and membrane movement at the plaque/glia interface. Microglia internalized systemically injected amyloid-binding dye at a much higher rate in the vicinity of plaques. These results indicate a role for microglia in plaque maintenance and provide a model with multiple targets for therapeutic intervention.

Hippocampal structural plasticity induced by entorhinal cortex (EC) lesions has been studied extensively in the rat, but little comparable research has been conducted in primates. In the current study we assessed the long-term effects of bilateral aspiration lesions of the EC on multiple markers of circuit organization in the hippocampal dentate gyrus of young adult monkeys (Macaca fascicularis). Alternate histological sections were processed for the visualization of somatostatin and vesicular acetylcholine transporter (VAChT) immunoreactivity and acetylcholinesterase histochemistry (AChE). The markers revealed the distinct laminar organization of dentate gyrus circuitry for stereology-based morphometric quantification. Consistent with findings in rats, the volume of the somatostatin-immunopositive outer molecular layer (OML), innervated by projections from the EC, was decreased by 42% relative to control values. The inner molecular layer (IML) displayed a corresponding volumetric expansion in response to denervation of the OML as measured by AChE staining, but not when visualized for quantification by VAChT immunoreactivity. Nonetheless, stereological estimation revealed a 36% increase in the total length of VAChT-positive cholinergic fibers in the IML after EC damage, along with no change in the OML. Together, these findings suggest that despite substantial species differences in the organization of hippocampal circuitry, the capacity for reactive plasticity following EC damage, previously documented in rats, is at least partly conserved in the primate dentate gyrus.

We have generated a novel transgenic mouse model on a C57BL/6J genetic background that coexpresses KM670/671NL mutated amyloid precursor protein and L166P mutated presenilin 1 under the control of a neuron-specific Thy1 promoter element (APPPS1 mice). Cerebral amyloidosis starts at 6-8 weeks and the ratio of human amyloid (A)beta42 to Abeta40 is 1.5 and 5 in pre-depositing and amyloid-depositing mice, respectively. Consistent with this ratio, extensive congophilic parenchymal amyloid but minimal amyloid angiopathy is observed. Amyloid-associated pathologies include dystrophic synaptic boutons, hyperphosphorylated tau-positive neuritic structures and robust gliosis, with neocortical microglia number increasing threefold from 1 to 8 months of age. Global neocortical neuron loss is not apparent up to 8 months of age, but local neuron loss in the dentate gyrus is observed. Because of the early onset of amyloid lesions, the defined genetic background of the model and the facile breeding characteristics, APPPS1 mice are well suited for studying therapeutic strategies and the pathomechanism of amyloidosis by cross-breeding to other genetically engineered mouse models.

The immediate-early gene (IEG) Arc is transcribed after behavioral and physiological treatments that induce synaptic plasticity and is implicated in memory consolidation. The relative contributions of neuronal activity and learning-related plasticity to the behavioral induction of Arc remain to be defined. To differentiate the contributions of each, we assessed the induction of Arc transcription in rats with fornix lesions that impair hippocampal learning yet leave cortical connectivity and neuronal firing essentially intact. Arc expression was assessed after exploration of novel environments and performance of a novel water maze task during which normal rats learned the spatial location of an escape platform. During the same task, rats with fornix lesions learned to approach a visible platform but did not learn its spatial location. Rats with fornix lesions had normal baseline levels of hippocampal Arc mRNA, but unlike normal rats, expression was not increased in response to water maze training. The integrity of signaling pathways controlling Arc expression was demonstrated by stimulation of the medial perforant path, which induced normal synaptic potentiation and Arc in rats with fornix lesions. Together, the results demonstrate that Arc induction can be decoupled from behavior and is more likely to indicate the engagement of synaptic plasticity mechanisms than synaptic or neuronal activity per se. The results further imply that fornix lesions may impair memory in part by decoupling neuronal activity from signaling pathways required for long-lasting hippocampal synaptic plasticity.

Major pathological findings in Alzheimer's disease (AD) brain include the deposition of amyloid-beta and synapse loss. Synaptic loss has been shown to correlate with the cognitive decline in AD patients, but the relationship between cerebral amyloidosis and synapse loss is complicated by the presence of neurofibrillary tangles and other lesions in AD brain. With the use of the APP23 transgenic mouse model that overexpresses human amyloid precursor protein (APP) with the Swedish double mutation, we investigated whether the development of cortical amyloid deposition was accompanied by synaptic bouton loss. With stereological methods, we show that despite robust age-related cortical amyloid deposition with associated synaptic degeneration, the total number of cortical synaptophysin-positive presynaptic terminals is not changed in 24-month-old animals compared with 3-, 8-, and 15-month-old APP23 mice. Wild-type mice also do not show an age-related loss of presynaptic boutons in the neocortex and are not significantly different from APP23 mice. Synaptophysin Western blotting revealed no significant difference between APP23 mice and wild-type controls at 3 and 25 months of age. Our results suggest that cerebral amyloidosis is not sufficient to account for the global synapse loss in AD. Alternatively, a putative trophic effect of APP may prevent, compensate, or delay a loss of synapses in this mouse model.

Alterations in the basal forebrain cholinergic system have been widely studied in brain aging and Alzheimer's disease, but the magnitude of decline and relationship to cognitive impairment are still a matter of debate. The rhesus monkey (Macaca mulatta) provides a compelling model to study age-related memory decline, as the pattern of impairment closely parallels that observed in humans. Here, we used antibodies against the vesicular acetylcholine transporter and a new stereological technique to estimate total cholinergic fiber length in hippocampal subregions of behaviorally characterized young and aged rhesus monkeys. The analysis revealed an age-related decline in the length of cholinergic fibers of 22%, which was similar across the hippocampal subregions studied (dentate gyrus granule cell and molecular layers, CA2/3-hilus, and CA1), and across the rostral-caudal extent of the hippocampus. This effect, however, was unrelated to performance on the delayed nonmatching-to-sample task, a test of recognition memory sensitive to hippocampal system dysfunction and cognitive aging in monkeys. These findings indicate that a decline in cholinergic input fails to account for the influence of normal aging on memory supported by the primate hippocampal region., (Copyright 2004 Wiley-Liss, Inc.)

Denervation of the dentate gyrus by entorhinal cortex lesion has been widely used to study the reorganization of neuronal circuits following central nervous system lesion. Expansion of the non-denervated inner molecular layer (commissural/associational zone) of the dentate gyrus and increased acetylcholinesterase-positive fibre density in the denervated outer molecular layer have commonly been regarded as markers for sprouting following entorhinal cortex lesion. However, because this lesion extensively denervates the outer molecular layer and causes tissue shrinkage, stereological analysis is required for an accurate evaluation of sprouting. To this end we have performed unilateral entorhinal cortex lesions in adult C57BL/6J mice and have assessed atrophy and sprouting in the dentate gyrus using modern unbiased stereological techniques. Results revealed the expected increases in commissural/associational zone width and density of acetylcholinesterase-positive fibres on single brain sections. Yet, stereological analysis failed to demonstrate concomitant increases in layer volume or total acetylcholinesterase-positive fibre length. Interestingly, calretinin-positive fibres did grow beyond the border of the commissural/associational zone into the denervated layer and were regarded as sprouting axons. Thus, our data suggest that in C57BL/6J mice shrinkage of the hippocampus rather than growth of fibres underlies the two morphological phenomena most often cited as evidence of regenerative sprouting following entorhinal cortex lesion. Moreover, our data suggest that regenerative axonal sprouting in the mouse dentate gyrus following entorhinal cortex lesion may be best assessed at the single-fibre level.

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.

The morphological changes that occur during normal brain aging are not well understood. This study used modern stereology to assess the effects of age and gender on total numbers of astrocytes and microglia in the hippocampal formation in C57Bl/6NNIA (B6) mice. Astrocytes and microglia were visualized using immunocytochemistry for glial fibrillary acidic protein (GFAP) and complement receptor 3 (Mac-1), respectively, and numbers of each cell type in dentate gyrus (DG) and CA1 regions were estimated using the optical fractionator method. The results reveal significantly greater ( approximately 20%) numbers of microglia and astrocytes in aged females compared to young female B6 mice. We also report that on average female B6 mice have 25-40% more astrocytes and microglia in DG and CA1 regions than age-matched male C57Bl/6J mice. Since astrocytes and microglia are thought to be targets of gonadal hormones, the effects of sex hormones and reproductive aging may be responsible for these findings.

Alzheimer's Disease (AD) is a neurodegenerative disorder that is characterized by extracellular deposits of amyloid-beta peptide (Abeta) and a severe depletion of the cholinergic system, although the relationship between these two events is poorly understood. In the neocortex, there is a loss of cholinergic fibers and receptors and a decrease of both choline acetyltransferase (ChAT) and acetylcholinesterase enzyme activities. The nucleus basalis of Meynert (NBM), which provides the major cholinergic input to the neocortex, undergoes profound neuron loss in AD. In the present study, we have examined the cholinergic alterations in amyloid precursor protein transgenic mice (APP23), a mouse model of cerebral beta-amyloidosis. In aged APP23 mice, our results reveal modest decreases in cortical cholinergic enzyme activity compared with age-matched wild-type mice. Total cholinergic fiber length was more severely affected, with 29 and 35% decreases in the neocortex of aged APP23 mice compared with age-matched wild-type mice and young transgenic mice, respectively. However, there was no loss of cholinergic basal forebrain neurons in these aged APP23 mice, suggesting that the cortical cholinergic deficit in APP23 mice is locally induced by the deposition of amyloid and is not caused by a loss of cholinergic basal forebrain neurons. To study the impact of cholinergic basal forebrain degeneration on cortical amyloid deposition, we performed unilateral NBM lesions in adult APP23 mice. Three to 8 months after lesioning, a 38% reduction in ChAT activity and significant cholinergic fiber loss were observed in the ipsilateral frontal cortex. There was a 19% decrease in Abeta levels of the ipsilateral compared with contralateral frontal cortex with no change in the ratio of Abeta40 to Abeta42. We conclude that the severe cholinergic deficit in AD is caused by both the loss of cholinergic basal forebrain neurons and locally by cerebral amyloidosis in the neocortex. Moreover, our results suggest that disruption of the basal cholinergic forebrain system does not promote cerebral amyloidosis in APP23 transgenic mice.

Quantification of linear biological structures has important applications in neuroscience; for example, the length of neurotransmitter-specific axonal innervation or length of dendritic processes within particular brain structures. Until recently, however, there have been practical limitations in the application of stereological tools for the unbiased estimation of object length on tissue sections. The recent development of efficient new approaches allows for the wider application of theoretically unbiased sampling and estimation techniques that are devoid of the assumptions and models of earlier methods. In this review, we outline the historical background and recent advances in the estimation of total length for biological objects on tissue sections, including a practical method to estimate the length of cholinergic fibers using newly developed methods. These newer methods also take advantage of three-dimensional image datasets and virtual probes, techniques that may have wider application in quantitative morphometry.

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.

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.

Quantification of linear biological structures has important applications in neuroscience; for example, the length of neurotransmitter-specific axonal innervation or length of dendritic processes within particular brain structures. Until recently, however, there have been practical limitations in the application of stereological tools for the unbiased estimation of object length on tissue sections. The recent development of efficient new approaches allows for the wider application of theoretically unbiased sampling and estimation techniques that are devoid of the assumptions and models of earlier methods. In this review, we outline the historical background and recent advances in the estimation of total length for biological objects on tissue sections, including a practical method to estimate the length of cholinergic fibers using newly developed methods. These newer methods also take advantage of three-dimensional image datasets and virtual probes, techniques that may have wider application in quantitative morphometry.

Cerebrovascular deposition of amyloid is a frequent observation in Alzheimer's disease patients. It can also be detected sporadically in normal aged individuals and is further found in familial diseases linked to specific gene mutations. The source and mechanism of this pathology are still unknown. It has been suggested that amyloidogenic proteins are derived from blood, the vessel wall itself, or from the central nervous system. In this article evidence is reviewed for and against each of these hypotheses, including new data obtained from transgenic mouse models. In APP23 transgenic mice that develop cerebral amyloid angiopathy (CAA) in addition to amyloid plaques, the transport and drainage of neuronally produced amyloid-beta (A beta) seem to be responsible for CAA rather than vascular A beta production or blood uptake. Although a number of mechanisms may contribute to CAA in humans, these results suggest that a neuronal source of A beta is sufficient to induce vascular amyloid deposition. The possibility to cross genetically defined mouse models of CAA with other mutant mice now has the potential to identify molecular mechanisms of CAA.

Transgenic mice that overexpress mutant human amyloid precursor protein (APP) exhibit one hallmark of Alzheimer's disease pathology, namely the extracellular deposition of amyloid plaques. Here, we describe significant deposition of amyloid beta (Abeta) in the cerebral vasculature [cerebral amyloid angiopathy (CAA)] in aging APP23 mice that had striking similarities to that observed in human aging and Alzheimer's disease. Amyloid deposition occurred preferentially in arterioles and capillaries and within individual vessels showed a wide heterogeneity (ranging from a thin ring of amyloid in the vessel wall to large plaque-like extrusions into the neuropil). CAA was associated with local neuron loss, synaptic abnormalities, microglial activation, and microhemorrhage. Although several factors may contribute to CAA in humans, the neuronal origin of transgenic APP, high levels of Abeta in cerebrospinal fluid, and regional localization of CAA in APP23 mice suggest transport and drainage pathways rather than local production or blood uptake of Abeta as a primary mechanism underlying cerebrovascular amyloid formation. APP23 mice on an App-null background developed a similar degree of both plaques and CAA, providing further evidence that a neuronal source of APP/Abeta is sufficient to induce cerebrovascular amyloid and associated neurodegeneration.

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.

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.

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.

Microglia are brain cells of considerable interest because of their role in CNS inflammatory responses and strong association with neuritic plaques in Alzheimer's disease (AD). In the present study, immunocytochemistry was combined with unbiased stereology to estimate the mean total number of microglia in dentate gyrus and CA1 regions of the mouse hippocampus. Systematic-uniform-random (SUR) sections were cut through the hippocampal formation of male C57BL/6J mice (n = 7, 4-5 months) and immunostained with Mac-1, an antibody to the complement subunit 3 receptor (CR3). The total number of Mac-1 immunopositive cells was determined using the optical fractionator method. The mean total number of microglia in the mouse dentate gyrus was estimated to be 20,300 (CV = 0.21) with a mean coefficient of error (CE) = 0.09. The mean total number of microglia in the mouse CA1 was estimated to be 43,200 (CV = 0.24) with a CE = 0.11. Comparison of total number estimates, derived from fraction- or volume-based methods, supported stereological theory regarding the equivalence of the two techniques. The time required to determine total microglia number in both hippocampal sub-regions was approximately 6 h per mouse from stained sections. The combination of immunocytochemistry and stereology provides a reliable means to assess microglia number that can easily be adopted for studies of transgenic and lesion-based models of aging and neurodegenerative diseases.

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.

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.

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid beta (A beta) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes A beta deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 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. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of A beta in the pathogenesis of the disease.

The present study provides an experimental model of the apoptotic death of pyramidal neurons in rat olfactory cortex after total bulbectomy. Terminal transferase (TdT)-mediated deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL), DNA electrophoresis, and neuronal ultrastructure were used to provide evidence of apoptosis; neurons in olfactory cortex were counted by stereology. Maximal TUNEL staining occurred in the piriform cortex between 18 and 26 hr postbulbectomy. Within the survival times used in the present study (up to 48 hr postlesion), cell death was observed exclusively in the piriform cortex; there was no evidence of cell death in any other areas connected with the olfactory bulb. Neurons undergoing apoptosis were pyramidal cells receiving inputs from, but not projecting to, the olfactory bulb. The apical dendrites of these neurons were contacted by large numbers of degenerating axonal terminals. Gel electrophoresis of DNA purified from lesioned olfactory cortex showed a ladder pattern of fragmentation. Inflammatory cells or phagocytes were absent in the environment of degenerating neurons in the early stages of the apoptotic process. The present model suggests that deafferentation injury in sensory systems can cause apoptosis. In addition, olfactory bulbectomy can be used for investigating molecular mechanisms that underlie apoptosis in mature mammalian cortical neurons and for evaluating strategies to prevent the degeneration of cortical neurons.

Development, ageing, and a variety of neurological disorders are characterized by selective alterations in specific populations of nerve cells which are, in turn, associated with changes in the numbers of synapses in the target fields of these neurons. To begin to delineate the significance of changes in synapses in development, ageing, and disease, it is first essential to quantify the number of synapses in defined regions of the CNS. In the past, investigators have used EM methods to assess synapse numbers or density, but these approaches are costly, labour intensive, and technically difficult, particularly in autopsy material. To begin to define reliable strategies useful for studies of both animals and humans, we used three techniques to measure synaptophysin-immunoreactivity in rat brain. The levels of synaptophysin protein were determined by Western blots of five hippocampal subregions; the intensity of synaptophysin-immunoreactivity in dentate gyrus and stratum oriens was determined by optical densitometry of immunocytochemically stained sections; and the total number of synaptophysin-immunoreactivity presynaptic boutons in dentate gyrus and stratum oriens was assessed by unbiased stereology. Each approach has advantages and disadvantages. Western blotting is the least time-consuming of the three methods and allows simultaneous processing of multiple samples. In systematically sampled histological sections, both densitometry and stereology allow precise definition of the region of interest, and the stereological optical dissector method allows quantitation of the numbers of synaptophysin-immunoreactive boutons. Stereology was the only method that clearly demonstrated greater synaptophysin-immunoreactivity in the dentate gyrus as compared to stratum oriens. The use of systematic sampling and the dissector technique offer a high degree of anatomical resolution (lacking in Western blot methods) and has quantitative advantage over the greyscale-based density approach. Thus, at present, stereology is the most useful method for estimating synaptic numbers in defined regions of the brain.