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The total number of cells--including both neurons and glial cells - was estimated in the neocortical part of the human fetal telencephalon in 22 normal brains within four major developmental zones: the cortical plate/marginal zone, the subplate, the intermediate zone and the ventricular/subventricular zone. The fetal ages ranged from 13 to 41 weeks of gestation. The cellular growth in the human fetal forebrain appears to be two-phased: one rapid, exponential phase from 13 to 20 weeks of gestation and a second and slower phase, which increases linearly, from approximately 22 weeks of gestation to term. From 13 to 20 weeks of gestation the total number of cells increases by a factor of 4.3 from 3 x 10(9) cells to 13 x 10(9) cells at 20 weeks of gestation. From mid-gestation to term, the total cell number increases by a factor of 2.9 to 38 x 10(9) cells in the newborn infant. Studying cellular growth in the normal human fetal brain is important since it may serve as a useful parameter for the assessment of cortical growth in non-invasive and histological studies, and thus improve the analysis of fetal brain disturbances.

The purpose of this study was to compare magnetic resonance imaging (MRI) against physical sectioning techniques to estimate the volume of human cerebral hemisphere compartments (cortex, subcortex, and their union, called "total"). The volume of these compartments was estimated postmortem for six human subjects from MRI virtual sections and from physical sections using the Cavalieri design with point counting. Cursory paired t tests revealed no significant differences between the two methods for any of the three compartments considered, although P = 0.06 for the subcortex. A sharper analysis incorporating recent error prediction formulae revealed a significant discrepancy between the two methods in the estimation of subcortex and total volume for three of the specimens. Yet, none of these analyses is adequate to detect possible biases. The incorporation of an explanatory variable, namely hemisphere weight, and the adoption of a specific gravity rho = 1.04 g/cm(3) for the material, enabled us to carry out an allometric analysis for the total compartment which revealed a significant bias of the MRI data. The new error prediction formulae are illustrated by way of example, and their accuracy is checked by a resampling experiment on a data set of 274 MRI sections.

Most studies agree that specific regions of the hippocampus and specific subcortical regions show neuronal loss in Alzheimer’s disease (AD). The aim of the study was to use design-based stereological methods to obtain an estimate of the total glial cell population in 14 AD cases and 20 controls to determine whether brains from AD patients have a different number of neocortical glial cells than controls. The mean total number of neocortical glial cells was 25.9 × 109 for the AD group and 29.1 × 109 for the control group, 2p = 0.18. The mean total number of neocortical neurons was 18.9 × 109 for the AD group and 21.2 × 109 for the control group, 2p = 0.059. Estimates of the sum of all glial cells and neurons in the neocortex were in the order of 50 billion cells with a glia to neuron ratio of 1.37 in both groups.

In a stereological study of the human substantia nigra (SN), the total number of melanin-positive and melanin-negative neurones from 28 male subjects aged 19-92 years was estimated using a uniform sampling design and optical disectors. There was a significant decrease in the total number of melanin-positive neurones as a function of age (r(2)=0.18, residual-CV=0.35, 2P=0.032). Using the rotator method, the size distribution of the melanin-positive neurones was estimated and showed a significant difference in mean cell volume of melanin-positive neurones between the seven youngest (21,077 microm(3)) and the seven oldest individuals (32,011 microm(3)), 2P=0.022. Using a combination of the total number of melanin-positive neurones and their size distribution, the total perikaryon volume of melanin-positive neurones could be estimated and showed no decrease with increasing age (r(2)=0.01, residual-CV=0.41, 2P=0.62). Age-related decline in dopamine-transporter neurones within the SN might explain the occurrence of extrapyramidal symptoms in many elderly individuals. Although age-related cell hypertrophy is usually considered to be an indication of cell degeneration or necrosis, this might not always be the case. The fact that motor symptoms, although present in many of the elderly, are of a limited nature despite the high percentage of lost neurones could be due to a compensatory increase in the cell body of dopamine-producing SN neurones. Thus, the total amount of cell substance capable of producing the essential transmitters might not be reduced to a critically low level as a result of ageing.

The previously shown wave of beta-cell apoptosis and the apparent plateau in the beta-cell mass in the third week of life in rats are still unexplained events. Using a novel design-based stereological method we investigated the postnatal development of the beta-cell population in Sprague-Dawley rats. The total beta-cell mass increased from postnatal day 4 until day 16, to be followed by a plateau until day 24, after which it increased further. This plateau was caused by beta-cell hypotrophia as well as decreased net beta-cell formation. The beta-cell mass per unit body weight (the relative beta-cell mass) was five times higher at birth compared with the adult constant level that was reached at approximately 24 days of age. We propose an explanatory model for the postnatal development of the beta-cell population in rats. According to this model, beta-cells in the early postnatal period are immature, i.e. are not susceptible to the mechanism that in later life maintains a constant relative beta-cell mass. Within the following weeks the number of mature beta-cells increases, and from approximately day 24 and onwards the beta-cell population is dominated by mature beta-cells that adjust to match the body weight, keeping a constant relative beta-cell mass. Findings of an apoptotic wave, a plateau phase in the total beta-cell mass development, a period with beta-cell hypotrophia, and the disappearance of insulin-like growth factor II positive beta-cells at postnatal day 21 all fit well in the model.

Kaplan, Suleyman/0000-0003-1477-5002; Canan, Sinan/0000-0002-9864-1767; pakkenberg, bente/0000-0003-3851-6494 WOS: 000174095200003 Seven days after 10 minutes bilateral occlusion of internal carotid arteries, the total neuron number and the volume of the hippocampus were estimated using the optical fractionator method for cell counting and Cavalieri's principle for volume estimation. After comparing with sham-operated animals, no significant difference in total neuron number was observed in hippocampi of ischemic chicks; a statistically significant increase in numerical density of hippocampal neurons in hippocampi of ischemic group (p

The neurobiological mechanisms underlying depression are not fully understood. Only a few previous studies have used validated stereological methods to test how stress and animal paradigms of depression affect adult hippocampal neurogenesis and whether antidepressant therapy can counteract possible changes in an animal model. Thus, in this study we applied methods that are state of the art in regard to stereological cell counting methods. Using a validated rat model of depression in combination with a clinically relevant schedule of electroconvulsive stimulation, we estimated the total number of newly formed neurons in the hippocampal subgranular zone. Also estimated were the total number of neurons and the volume of the granule cell layer in adult rats subjected to chronic restraint stress and electroconvulsive stimulation either alone or in combination. We found that chronic restraint stress induces depression-like behavior, without significantly changing neurogenesis, the total number of neurons or the volume of the hippocampus. Further, electroconvulsive stimulation prevents stress-induced depression-like behavior and increases neurogenesis. The total number of neurons and the granule cell layer volume was not affected by electroconvulsive stimulation.

Our knowledge of the relationship between brain structure and cognitive function is still limited. Human brains and individual cortical areas vary considerably in size and shape. Studies of brain cell numbers have historically been based on biased methods, which did not always result in correct estimates and were often very time-consuming. Within the last 20–30 years, it has become possible to rely on more advanced and unbiased methods. These methods have provided us with information about fetal brain development, differences in cell numbers between men and women, the effect of age on selected brain cell populations, and disease-related changes associated with a loss of function. In that this article concerns normal brain rather than brain disorders, it focuses on normal brain development in humans and age related changes in terms of cell numbers. For comparative purposes a few examples of neocortical neuron number in other mammals are also presented.

The total number of neurons and glial cells in the mediodorsal thalamic (MDT) nucleus of four aged females with Down syndrome (DS; mean age 69years) was estimated and compared to six age- and sex-matched controls. The MDT nucleus was delineated on coronal sections, and cell numbers (large and small neurons, oligodendrocytes, and astrocytes) were estimated using the optical fractionator technique. The DS brains had an average of 3.41×10(6) total neurons in the MDT nucleus in contrast to 5.97×10(6) in the controls, with no overlap (2p=0.004), affecting large (projecting) and small (local inhibitory) neurons nearly equally. In contrast, we observed no significant differences in either glial cell population. The cortical structures of the same four DS brains were previously estimated to be half the normal size of controls with a reduction in cell numbers whereas the basal ganglia were unaffected. As DS brains are affected by developmental delay, premature aging, and Alzheimer-like pathology, the finite cause of the reduced number of cells in MDT nucleus cannot be determined; however, these findings provide stereological evidence for a local reduction in neuron numbers in the MDT nucleus, which could affect the cognitive capacity of patients with DS.

To understand the dynamics of islet population, especially during conditions with growth of the total islet mass, it is important to have reliable estimators of parameters describing the quantitative appearance of the islet population. We describe a stereological estimator of the volume-weighted mean islet volume based on unbiased assumption-free stereological principles. The volume-weighted mean islet volume is the mean islet volume if the islets are weighted (sampled) proportional to their volume. This method allows simultaneously unbiased estimation of the total islet mass. With use of this method, 22 male Sprague-Dawley rats within the age span of 34–102 days old were investigated. We found a linear correlation (P < 0.001) between total islet mass and the volume-weighted mean islet volume. The results support models demonstrating that the physiological growth of the total islet mass in the period studied is totally or mainly caused by proportional growth of existing islets. The functional meaning of the volume-weighted mean islet volume is discussed, and previous methods to study the mean islet volume and islet number are critically evaluated. We propose the volume-weighted mean islet volume to be a biologically useful parameter when describing the mean volume of the pancreatic islets and investigating the differences between experimental groups.

The aim of the present study was to estimate mean neuronal volume and absolute size distributions of the neocortical neurons in brains from controls and AD patients using stereological methods based on unbiased principles to determine whether changes in absolute cell size are part of the neuropathological pattern of Alzheimer's disease. The neocortex of 8 patients with Alzheimer's disease (AD), mean age 81.1 (68-94) y was compared with 9 nondemented controls, mean age 80.9 (65-101) y. The brains came from Johns Hopkins University Hospital (JHUH) in Baltimore, USA, the Netherlands Brain Bank (NBB), and from a large brain repository in Denmark. The rotator method was used to obtain an estimate of cell volumes providing absolute size distributions of the volume of both cell perikaryon and cell nuclei. The geometric mean volume of cell nuclei in neocortical neurons was 328 microm3 (interindividual CV = 0.15) in the Alzheimer group compared with 277 microm3 (interindividual CV = 0.17) in controls which was a statistically significant increase (P = 0.049). The perikaryal volume was 1117 microm3 in the Alzheimer group compared with 999 microm3 in controls which was a nonsignificant difference (P = 0.20). There was a highly significant correlation between the nuclear and perikaryal volumes in all individuals. The average slope of the regression lines was significantly higher in the Alzheimer patients than in the controls, illustrating that nuclear hypertrophy was more pronounced in the largest neurons.

Stereological studies have contributed with important results to the understanding of brain abnormalities in schizophrenia. The data obtained from stereological studies of brains from schizophrenic patients, including studies of the thalamus, hippocampus, and cortex, are discussed and suggest a central role of the thalamic nuclei in the etiology of the disease. The basic stereological tools are presented and possible biases in quantification studies are discussed.

We describe a method for unbiased assumption-free estimation of the total number of beta-cells and the mean beta-cell volume in mouse and rat pancreas based on light microscopy. Such a method, which takes advantage of one of the most recent developments in stereology, the fractionator, has not previously been described. It relies on repeated fractionation of the tissue using systematic uniform random sampling combined with an unbiased counting principle. The method was applied to eight BALB/cBom male mice (56 days) and six Lewis/MOL male rats (47 days). In mice, the total number of beta-cells was 1.06 +/- 0.07 x 10(6) (mean +/- SEM) per pancreas with a mean beta-cell volume of 1280 +/- 17 microm3, while in rats the total beta-cell number was 2.76 +/- 0.42 x 10(6) per pancreas with a mean beta-cell volume of 1170 +/- 65 microm3. Furthermore, the results showed that in both species the biological variability in the total beta-cell volume is due to differences in the number of beta-cells rather than variability of the mean beta-cell volume. The method can be used to give a precise description of number and volume of beta-cells at different ages, and will make it possible to estimate the contributions of hyper/hypotrophia and hyper/hypoplasia to a given induced or spontaneous change in the total beta-cell mass.

The aim of the present study was to estimate the volume of the ventricular system comprising lateral plus third ventricles in patients with Gilles de la Tourette's syndrome on computed tomographic (CT) scannings using unbiased stereological principles and to compare that volume with a control group. We found a significantly reduced ventricular volume in 24 patients with Gilles de la Tourette's syndrome (GTS) compared with 28 controls.

Autologous platelet-rich fibrin (PRF(®)) is prepared by the automatic Vivostat(®) system. Conflicting results with Vivostat PRF in acute wound healing prompted us to examine its cellular and biomolecular composition. Specifically, platelets, selected growth factors and matrix metalloproteinase (MMP)-9 were quantified using novel analytical methods.Ten healthy non-thrombocytopenic volunteers donated blood for generation of intermediate fibrin-I and final PRF. Anticoagulated whole blood and serum procured in parallel served as baseline controls. Leucocyte, erythrocyte and platelet counts in whole blood and fibrin-I were determined by automated haematology analyser. Platelet concentration in PRF was quantified manually by stereologic analysis of Giemsa-stained tissue sections, and the total content of five growth factors and MMP-9 by enzyme-linked immunosorbent assays.The number of leucocytes and erythrocytes was reduced (P0·001), whereas platelets increased (P0·001) in fibrin-I versus whole blood. PRF contained 982 ± 206 × 10(9) platelets/l representing 3·9-fold (P0·001) enrichment relative to whole blood. Growth factor abundance in Vivostat PRF and serum was in descending order: transforming growth factor-β1 [5·1-fold higher in PRF than serum, P0·001]platelet-derived growth factor (PDGF)-AB [2·5-fold, P0·01]PDGF-BB [1·6-fold, P0·05]vascular endothelial growth factorbasic fibroblast growth factor [75-fold, P0·001]. MMP-9 was reduced 139-fold (P0·001) compared with serum, reflecting leucocyte depletion in PRF.The gained knowledge on platelet enrichment and biomolecular constituents may guide clinicians in their optimal use of Vivostat PRF for tissue regenerative applications.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder causing marked pathology in the motoneuron system. The pathophysiology of the selective degeneration of motor neurons in the disease is as yet unknown, but evidence suggests that excitotoxic mechanisms might be involved. The present study was undertaken to determine whether defects in neurotransmitter receptors are involved in the disease, analyzing uniformly sampled specimens from neocortex and motorcortex. The binding to benzodiazepine, muscarinic cholinergic, and NMDA receptors in ALS brains was compared to that in control brains, using a single radioligand concentration of [3H]Ro 15-1788, [3H]QNB and [3H]MK-801. The benzodiazepine and the muscarinic cholinergic receptor binding was unaffected in any cortical region from the ALS subjects compared to controls. NMDA receptor binding labeled by [3H]MK-801 was significantly increased in several neocortical regions in the ALS group compared to the control group. Scatchard analysis of [3H]MK-801 binding in frontal cortex revealed a single binding site with an unaltered maximal binding capacity but an increased binding affinity of the site in the ALS group compared to the controls. The generalized alteration in the affinity of the binding site for [3H]MK-801 in the ALS cortex may indicate a modification of the NMDA receptor due to different sensitivity for endogenous modulators or to a different subunit composition of the NMDA receptor in ALS with altered functional properties. These findings may reflect a pathophysiological phenomenon in ALS.

Decreased parvalbumin expression is a hallmark of the pathophysiology of schizophrenia and has been associated with abnormal cognitive processing and decreased network specificity. It is not known whether this decrease is due to reduced expression of the parvalbumin protein or degeneration of parvalbumin-positive interneurons (PV(+) interneurons). In this study, we examined PV(+) expression in two rat models of cognitive dysfunction in schizophrenia: the environmental social isolation (SI) and pharmacological neonatal phencyclidine (neoPCP) models. Using a stereological method, the optical fractionator, we counted neurons, PV(+) interneurons, and glial cells in the medial prefrontal cortex (mPFC) and hippocampus (HPC). In addition, we quantified the mRNA level of parvalbumin in the mPFC. There was a statistically significant reduction in the number of PV(+) interneurons (p = 0.021) and glial cells (p = 0.024) in the mPFC of neonatal phencyclidine rats, but not in SI rats. We observed no alterations in the total number of neurons, hippocampal PV(+) interneurons, parvalbumin mRNA expression or volume of the mPFC or HPC in the two models. Thus, as the total number of neurons remains unchanged following phencyclidine (PCP) treatment, we suggest that the decreased number of counted PV(+) interneurons represents a reduced parvalbumin protein expression below immunohistochemical detection limit rather than a true cell loss. Furthermore, these results indicate that the effect of neonatal PCP treatment is not limited to neuronal populations.

The Cavalieri and Vertical Sections methods of design based stereology were applied in combination with 3 tesla (i.e. 3T) Magnetic Resonance Imaging (MRI) to estimate cortical and subcortical volume, area of the pial surface, area of the grey-white matter boundary, and thickness of the cerebral cortex. The material comprises eight human cadaveric cerebri which had been separated into sixteen cerebral hemisphere specimens prior to embedding in agar gel. The results from MRI were compared with corresponding 'gold standard' values subsequently obtained by application of the same methodology using physical sectioning of the specimens. 95% agreement intervals revealed poor agreement between MR imaging and physical sectioning, specially for pial surface and thickness, as well as cerebral cortex and subcortex volumes. On average, pial surface area was estimated to be almost half the extent using MRI compared to physical sectioning (i.e. 45%, p

An increasing number of people are living past the age of 100 years, but little is known about what differentiates centenarians from the rest of the population. In this study, brains from female subjects in 3 different age groups, 65-75 years (n = 8), 76-85 years (n = 8), and 94-105 years (n = 7), were examined to estimate the total number of neocortical neurons, astrocytes, oligodendrocytes, and microglia. There was no statistically significant difference in the mean number of neocortical neurons between the 3 groups: 17.9 × 10(9) (CV = SD/mean = 0.15) in the youngest group, 18.1 × 10(9) (CV = 0.22) in the second group, and 16.32 × 10(9) (CV = 0.24) in the oldest group. However, there was a significant difference in the total number of neocortical glial cells between the youngest (41.0 × 10(9)) and oldest (29.0 × 10(9)) age groups (p = 0.013). The significance was probably driven by a significant difference in the total number of neocortical oligodendrocytes that differed significantly between the youngest (27.5 × 10(9)) and oldest (18.1. × 10(9), p = 0.006) age groups. In conclusion, very old individuals have brain neuron numbers comparable with younger individuals, which may be encouraging for those who live into the "fourth age" and may contribute to the longevity of this exceptional group of people.

Objectives The aim of the present randomized clinical study was to evaluate histologically whether the addition of cultivated, autogenous bone cells to a composite graft of deproteinized bovine bone mineral (DBBM) and autogenous bone (AB) for sinus floor augmentation (SFA) enhance bone formation compared with what achieved after SFA with DBBM + AB alone. Material and methods Twenty patients with remaining posterior maxillary alveolar crest height of less than 3 mm received SFA after randomization either with an DBBM and AB composite in a 1 : 1 ratio or with DBBM + AB supplemented with autogenous bone cells, which were cultivated from a bone biopsy harvested earlier from the tuberosity area. Four months after SFA, two cylindrical biopsies were taken from the augmented sinuses concomitantly with the implant site preparation by means of a trephine bur. An additional biopsy was taken from the tuberosity area. Bone density at the augmented sinus and the tuberosity area and the height of augmentation were estimated on non-decalcified histological sections prepared from the biopsies. A relative bone density index (RBD) was also calculated by dividing bone density at the augmented sinus with bone density at the tuberosity area. Results All patients but one could receive two implants after SFA; in one patient, only one implant could be placed. All implants were osseointegrated and could be loaded. Median bone density in the sinus was 30% and 25% in the cell seeded and no-cells added DBBM + AB groups, respectively. Bone augmentation height averaged 6.0 and 5.4 mm and RBD averaged 0.48 and 0.73 in the cell seeded and no-cells added DBBM + AB groups, respectively. None of the differences between groups was statistically significant. Conclusions Cultivated autogenous bone cell seeded to a DBBM + AB composite did not significantly improve bone formation (density and height) after SFA, compared with what was achieved with DBBM + AB alone. Both approaches resulted into enough bone to support implant placement and osseointegration.

Background Stereology is the study of estimating geometric quantities. When successfully applied, the combination of immunohistochemistry (IHC) and stereology eliminates intra- and interobserver variability for cell type identification. Methodology/Principal Findings We propose a method to validate existing antibody based cell type markers for stereological application. Comparison was made on the 100-days-old Gottingen minipig (G-mini) neocortex between estimates of total neuron number derived from Giemsa staining using morphological criteria and immunohistochemistry-based cell counting with NeuN. The mean total neuron numbers estimated by the two staining methods were not significantly different. Estimated quantities, including glial cell number, neocortical volume, cell densities and glial-to-neuron ratio were also presented. Additionally, we assessed other commonly used glial markers and discussed how to evaluate the advantages and disadvantages of these markers for stereological estimation of cell number. Conclusion/Significance The concordance in quantitative estimates of total neuron number derived from NeuN- and Giemsa-stained sections provides evidence for the sensitivity and specificity of NeuN as a neuronal marker in the G-mini. Although time-consuming, quantitative validation of IHC should always be considered in stereological studies if there is doubt of the sensitivity, specificity, or reproducibility of cell type markers. Inaccurate staining may cause both over- and underestimation of the total cell number and inflict considerable limitation when analyzing the results.

Precise estimates of total neuron numbers in neocortices of 11 women, mean age 82.6 years (range 79-88) with severe senile dementia of the Alzheimer's type (SDAT) were compared with similar estimates in 10 cognitively normal women of comparable mean age (84.1 years; range 74-92). The total mean nerve cell number in the SDAT group was 16.9 x 10(9) with a coefficient of variation (CV = SD/mean) = 0.14, whereas mean total neuron number in the control group was 18.1 x 10(9), CV = 0.18. In a material of this size the reduction of 6% in neocortical cell number in the SDATs is neither statistically nor biologically significant. Nevertheless, all patients with SDAT were severely demented, having a mean score of 5.6 on a 1-7-scale of dementia. This contrasts with the nondemented individuals who had lived an independent life at home until shortly before death. The SDAT patients showed a rather consistent reduction in cortical volume by 14%, an atrophy that was solely due to a reduced cortical thickness. In addition, all had multiple neocortical plaques (Bielschowsky silver stain).

Our aim was to investigate a model of the morphologic approach proposed in guidelines for developmental neurotoxicity testing (DNT). Hereto, a limited DNT study [EPA Health Effects Test Guidelines OPPTS 870.6300, 1996a. Developmental Neurotoxicity Study "Public Draft", United States Environmental Protection Agency; Prevention, Pesticides and Toxic Substances (7101); EPA 712-C-96-239, June 1996. http://www.epa.gov/opptsfrs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Drafts/870-6300.pdf was carried out with different doses of methylazoxy methanol acetate (MAM), known to affect brain morphology and neuron numbers in the developing brain. After gross examination, the brains of F1-animals were further dissected along neuro-anatomical landmarks to ensure homology between tissues of different individuals. The (relative) weight of the brain (parts) was determined. One brain half (alternating left/right to avoid lateralization) was further used for microscopic slide reading and measurement of brain layer width (linear morphometry); the other was set aside for stereologic investigation in a later phase of the study. In the offspring, a clear reduction in brain size (gross macroscopy) and weight (MAM high- and top-dose groups) was observed on postnatal days (PN) 22 and 62, but this reduction was hard to pinpoint in the microscope as the changes primarily appeared quantitative in nature, rather than qualitative. Linear measurements of brain layer width appeared very sensitive and efficient. This first step of a project is presented and the perspectives of a further stereologic investigation are discussed. © 2005 Published by Elsevier B.V.

The Göttingen minipig (G-mini) is increasingly used as a non-primate model for human neurological diseases. We applied design-based stereology on five groups of G-minis aged 1 day, 14 days, 30 days, 100 days, and 2 years or older to estimate the pattern of postnatal neuron number development in the neocortex. Two time periods for the postnatal increase of neocortical neuron number were observed from the time of birth to day 14 (P=0.013) and from day 30 to day 100 (P0.001). No significant change in neuron number was found from day 14 to 30 (P=0.58) and day 100 onward (P=0.39). The average estimated total number of neurons in the neocortex was 236, 274, 264, 338, and 353 million, respectively. Since neurogenesis and neuronal migration in the human neocortex are generally accepted to be complete before term, the application of G-mini as human disease models may be inappropriate before day 100. However, G-mini may serve as a valuable model for the studies of ongoing neurogenesis in the living brain.

The total numbers of neurons and glial cells in the neocortex and basal ganglia in adults with Down syndrome (DS) were estimated with design-based stereological methods, providing quantitative data on brains affected by delayed development and accelerated aging. Cell numbers, volume of regions, and densities of neurons and glial cell subtypes were estimated in brains from 4 female DS subjects (mean age 66 years) and 6 female controls (mean age 70 years). The DS subjects were estimated to have about 40% fewer neocortical neurons in total (11.1 × 10(9) vs. 17.8 × 10(9), 2p ≤ 0.001) and almost 30% fewer neocortical glial cells with no overlap to controls (12.8 × 10(9) vs. 18.2 × 10(9), 2p = 0.004). In contrast, the total number of neurons in the basal ganglia was the same in the 2 groups, whereas the number of oligodendrocytes in the basal ganglia was reduced by almost 50% in DS (405 × 10(6) vs. 816 × 10(6), 2p = 0.01). We conclude that trisomy 21 affects cortical structures more than central gray matter emphasizing the differential impairment of brain development. Despite concomitant Alzheimer-like pathology, the neurodegenerative outcome in a DS brain deviates from common Alzheimer disease.

The red-eared turtle is an important animal model for investigating the neural activity in the spinal circuit that generates motor behavior. However, basic anatomical features, including the number of neurons in the spinal segments involved, are unknown. In the present study, we estimate the total number of neurons in segment D9 of the spinal cord in the red-eared turtle (Trachemys scripta elegans) using stereological cell counting methods. In transverse spinal cord sections stained with modified Giemsa, motoneurons (MNs), interneurons (INs), and non-neuronal cells were distinguished according to location and morphology. Each cell type was then counted separately using an optical disector with the cell nucleus as counting item. The number of cells in segment D9 was as follows (mean ± SE): MNs, 2049 ± 74; INs, 16,135 ± 316; non-neuronal cells, 47,504 ± 478 (n= 6). These results provide the first estimate of the total number of neurons in a spinal segment in a terrestrial vertebrate based on unbiased stereological methods and an upper bound on the number of neurons involved in segmental sensorimotor activity. These findings also form a crucial quantitative foundation for integrating electrophysiological data into mathematical circuit models.

The total number of neurons was estimated in eight brains from chronic schizophrenic men and compared with 16 gender- and age-matched controls. The average total neocortical nerve cell number was 22.12·10 9 in schizophrenics and 22.06·10 9 in controls. The estimate of total cell numbers can not demonstrate more subtle neuronal losses of specific cell types or cell loss in specific regions, but the result does not indicate that a major cell loss in the neocortex of schizophrenics is part of the disease. The sampling scheme was based on a uniform sampling design, the counting was performed using new stereological principles and the majority of the cerebral cortex was left intact providing the possibility for resampling and further analysis.

Although it is commonly believed that chronic alcohol abuse results in loss of neocortical neurons, this assumption has not been properly tested. We used new stereological techniques to make a precise and unbiased estimate of the total number of neurons in the neocortex of brains obtained at necropsy from 11 chronic alcoholic men and 11 control men. The groups were matched with respect to age and height. Total mean neocortical neuron numbers in the two groups did not differ (alcoholics 23.4 x 10(9), controls 23.2 x 10(9)). Estimation of macroscopic brain volumes showed significant reductions in alcoholics compared with controls of the volume/weight ratios of white matter (11%, p = 0.013) and of archicortex (30%, p = 0.028). The volume of the ventricles in the alcoholic group was enlarged by 26%, but this was not statistically significant. There was no difference in the volumes of the neocortices. Our study confirms that chronic alcoholics lose white matter, and this could provide the basis for their functional impairment. However, the results also suggest that the observed brain damage in the alcoholic group is potentially reversible since preserved nerve-cell bodies might allow lost or malfunctioning axons to re-established and restored to function after prolonged abstinence and/or treatment. By contrast, lost neocortical neurons cannot be replaced.

During aging, decline in memory and cognitive abilities as well as motor weakening is of great concern. The dopaminergic system mediates some aspects of manual dexterity, in addition to cognition and emotion, and may be especially vulnerable to aging. A common neurodegenerative disorder of this system, Parkinson's disease, is characterized by a selective, progressive loss of dopaminergic neurons in the substantia nigra pars compacta. This review includes studies quantifying age and Parkinson's-related changes of the substantia nigra, with emphasis on stereological studies performed in the substantia nigra pars compacta.

The development of the human neocortex is a complex and highly regulated process involving a time-related expression of many transcription factors including the homeobox genes Pax6 and Meis2. During early development, Pax6 is expressed in nuclei of radial glia cells in the neocortical proliferative zones and controls the differentiation and neurogenetic fate of these cells in the dorsal telencephalon in rodents. Animal studies on the Meis2 gene have revealed expression in the developing telencephalon and Meis2 is known to regulate the expression of Pax6 in the eye and pancreas. Because of this functional relation between Pax6 and Meis2, we studied the spatial and temporal expression of PAX6, and MEIS2 using a developmental series of human fetal brains at 7-19 postconceptional weeks with emphasis on the forebrain to investigate whether the two genes are expressed in the same regions and zones in the same time window. We demonstrate by in situ hybridization and immunohistochemistry that the two homeobox genes are expressed during early fetal brain development in humans. PAX6 mRNA and protein were located in the proliferative zones of the neocortex and in single cells in the cortical preplate at 7 fetal weeks and in the developing cortical plate from 8 or 9 to 19 fetal weeks. The expression of PAX6 expanded into the ganglionic eminence just prior to the stage at which a stereological estimation showed an exponential rise in total cell number in this area. The MEIS2 gene was also present in the proliferative zones of the human fetal neocortex and a higher expression of MEIS2 than PAX6 was observed in these areas at 9 fetal weeks. Further, MEIS2 was expressed at a very high level in the developing ganglionic eminence and at a more moderate level in the cortical plate.

Estimates of total number of different neuron and glial cell types in the dentate nuclei and the four major regions of the human cerebellum were obtained by new stereological methods. With the optical disector and Cavalieri methods, the total number of neurons and glial cells in the human cerebellum of five elderly men was estimated to be 105,000 × 106 (coefficient of variation, CV = 0.13). Of this total, the granule cells comprised 101,000 × 106 (CV = 0.13) and the Purkinje cells comprised 30.5 × 106 (CV = 0.13) of the neurons. The mean of the total number of neurons in the dentate nucleus was 5.01 × 106 (CV = 0.28). The average surface area of the human cerebellum was estimated to be 1160 cm2 (CV = 0.29). The rationale for the different sampling schemes used to quantify the various cell types is described. © 1992 Wiley-Liss, Inc.

Reductions of 40% in total cell number and 25% in volume of the mediodorsal thalamic nucleus were recently reported in an unbiased neurostereological study of neuroleptic-treated schizophrenic patients. In order to investigate whether these results might be secondary to many years of treatment with neuroleptic drugs, eight brains from schizophrenics never treated with neuroleptics and eight controls were studied using the unbiased Cavalieri volume estimator. To compare left-right differences in this region, twelve neuroleptic-treated schizophrenics and eleven control cases were compared. The brains used for the left-right comparison study and five of 20 used for comparison of treated and untreated brain volumes have been used in an earlier study. The mediodorsal thalamus volume was reduced by 31% in untreated schizophrenics and by 22% in neuroleptic-treated schizophrenics. No differences were found in mean total volume of the left and right mediodorsal thalamus in brains from controls nor from schizophrenics. A major difference exists with respect to time of fixation in controls (12 years) and untreated schizophrenics (39 years) that makes shrinkage differences a possible confounding variable. The results suggest that the consistent reduction in number of neurons in the mediodorsal thalamic nucleus are not secondary to prolonged treatment with neuroleptic drugs and that asymmetry in this specific brain region is not a feature of the schizophrenia-afflicted brain.

Nonquantitative studies indicate that the cerebellum is neuropathologically affected in Alzheimer's disease; however, no quantitative studies on the subject have yet been conducted. Ten cerebella from elderly female subjects with severe Alzheimer's disease and 10 age- and gender-matched controls were examined. The cerebellum was divided into 5 regions and the Purkinje and granule cell number and density, cortical volume, molecular and granular layer volume and thickness, white matter volume, surface area, and the Purkinje cell gradient were stereologically estimated. There was no significant difference between the groups in Purkinje or granule cell number or density, and no overall difference in Purkinje cell gradient. However, there was a significant 12.7% reduction in total cerebellar volume in the Alzheimer's group and significant localized differences between the groups regarding other parameters. The relative lack of neuropathological changes in the cerebellum of severely demented Alzheimer's patients suggests that neuronal cell bodies on a global scale apparently still are intact.

Stereology is a set of mathematical and statistical tools to estimate three-dimensional (3-D) characteristics of objects from regular two-dimensional (2-D) sections. In medicine and biology, it can be used to estimate features such as cell volume, cell membrane surface area, total length of blood vessels per volume tissue and total number of cells. The unbiased quantification of these 3-D features allows for a better understanding of morphology in vivo compared with 2-D methods. This review provides an introduction to the field of stereology with specific emphasis on the application of stereology to dermatological research by supplying a short insight into the theoretical basis behind the technique and presenting previous dermatological studies in which stereology was an integral part. Both the theory supporting stereology and a practical approach in a dermatological setting are reviewed with the aim to provide the reader with the capability to better assess papers employing stereological estimators and to design stereological studies independently.

Using an unbiased stereological technique, the total numbers of pigmented and non-pigmented neurons were estimated in the substantia nigra of seven patients with Parkinson's disease and seven control patients. Compared with the controls, in which the average total number of pigmented neurons was 550,000, the number of neurons was reduced by 66% in the patients. The average total number of non-pigmented neurons was 260,000 in controls and reduced by 24% in the patients. A significant correlation (r = 0.81) existed between the total numbers of pigmented and non-pigmented neurons in the controls, whereas a similar correlation (r = 0.72) in the patients fell just short of statistical significance. The stereological estimates made in this study are unbiased, in that they are independent of nerve cell size, section thickness and of dimensional changes in brain tissue induced by histological procedures. The stereological method is considerably more efficient than previous conventional methods.

White matter changes have been reported as part of Alzheimer dementia. To investigate this, the total subcortical myelinated nerve fiber length was estimated in postmortem brains from eight females (age 79–88 years) with severe Alzheimer's disease (AD) and compared with brains from 10 female control subjects (age 74–92 years). A stereological method for estimating myelinated brain fibers includes sampling systematically, randomly from the white matter, and counting fibers in unbiased counting frames using light microscopy at approximately 6000× magnification. The diameter of each counted fiber was measured to obtain the diameter distribution of myelinated fibers in both groups. The mean total myelinated fiber length was 81,554 km in the AD group and 78,896 km in the control group ( P =0.63). All other measured parameters were also unaffected in the AD brains: The mean fiber length density was 248 km/cm 3 in the AD group and 247 km/cm 3 in the control group; the volume of white matter was 329 cm 3 (AD) and 321 cm 3 (control) and the volume density of myelinated fibers to white matter tissue volume was 0.30 in AD group and 0.31 in the control group. This is the first study of subcortical brain white matter fiber length using a stereological method on postmortem brains from AD patients and control subjects.

Several stereological studies of schizophrenic subjects have shown reduction in both the total number of neurons and in the total volume of the mediodorsal thalamic nucleus (MD). This is in contrast to other studies in that no differences have been found. Using systematic random sampling and an optical fractionator design, the total number of neuron and glial cells in the MD subdivisions: parvocellular (MDPC), magnocellular (MDMC), and densocellular (MDDC) were counted in brains from 9 schizophrenic and 8 control subjects. The control subjects were age, height and body-weight matched to the schizophrenic subjects. We found the neuronal numbers in the schizophrenic subjects to range more than a factor of two, from 3.68 to 9.22 x 106. This is in contrast to the control subjects, who ranged from 5.24 to 7.10 x 106 in neuronal cell numbers. Within our inhomogeneous sample, some schizophrenic subjects thus exhibited relative high total neuron numbers in MD, while others exhibited relative low neuron numbers. The result is in line with the heterogeneity of this severe mental disease and may help to explain why different research groups get different results. The major limitation in this study is the small number of brains of schizophrenic subjects with a high degree of inhomogeneity in length of disease and age of onset. The debates of the comparison of the neurons in the MD in brains of schizophrenic subjects and control subjects and the possible impact of this variance on the disease are still not complete.

An efficient method for the unbiased estimation of the total number of neurons in rat brain cortex is presented. The method is reasonable fast, the counting procedure takes 2-3 h per rat, and gives an estimate of the total neuron number in cortex cerebri with an CE of 0.10. The rat cerebral cortex is found to have a mean volume of 253 mm3 and to contain a mean of 21 million neurons. The method has been used in a neurotoxicologic study, in which rats were given toluene perorally in doses of 200, 400 and 800 mg/kg/day, respectively, for 12 weeks, followed by an exposure-free period of 4 weeks. The total number of neurons in rat brain cortex cerebri showed no significant difference between exposed and non-exposed animals. The applicability of the method is discussed.

Udgivelsesdato: 2007-Jul The psychostimulant 3,4-methylendioxymethamphetamine (MDMA, "ecstasy") evokes degeneration of telencephalic serotonin innervations in rodents, nonhuman primates, and human recreational drug users. However, there has been no alternative to nonhuman primates for studies of the cognitive and neurochemical consequences of serotonin depletion in a large-bodied animal. Therefore, we used positron emission tomography (PET) with [(11)C]DASB to map the distribution of plasma membrane serotonin transporters in brain of Göttingen minipigs, first in a baseline condition, and again at 2 weeks after treatment with MDMA (i.m.), administered at a range of doses. In parallel PET studies, [(11)C]WAY-100635 was used to map the distribution of serotonin 5HT(1A) receptors. The acute MDMA treatment in awake pigs evoked 1 degrees C of hyperthermia. MDMA at total doses greater than 20 mg/kg administered over 2-4 days reduced the binding potential (pB) of [(11)C]DASB for serotonin transporters in porcine brain. A mean total dose of 42 mg/kg MDMA in four animals evoked a mean 32% decrease in [(11)C]DASB pB in mesencephalon and diencephalon, and a mean 53% decrease in telencephalic structures. However, this depletion of serotonin innervations was not associated with consistent alterations in the binding of [(11)C]WAY-100635 to serotonin 5HT(1A) receptors. Stereological cell counting of serotonin-positive neurons, which numbered 95,000 in the dorsal raphé nucleus of normal animals, was unaffected in MDMA-treated group. group.

The rhesus monkey is widely used as an experimental animal model in the study of brain function and disease. While previous quantitative studies have provided knowledge of regional numbers, little is known of the total neocortical neuron and glial cell numbers in this species. The aim of this study is to establish quantitative norms. We use the optical fractionator and Cavalieri principle to examine the right hemisphere of eight young rhesus monkeys taken from the control group of an ongoing study. Applying these methods to agar-embedded and vibratome-sectioned tissue, we generate estimates of cell numbers and regional volumes of neocortical and hippocampal regions with coefficients of variance (CV) around 10%. The mean unilateral neocortical neuron number is 1.35 × 109 (CV ± 0.10) and the mean unilateral neocortical glial cell number is 0.78 × 109 (CV ± 0.17). Mean unilateral neocortical volume is found to be 8.5 (CV ± 0.10) cm3 after processing, or 19 cm3 when correcting for shrinkage. The neuron/glia ratio is 1.77. The neurons are distributed with 18% in the frontal cortex, 57% in the temporal and parietal cortices, and 25% in the occipital cortex. In the hippocampal subregions, we found unilateral neuron number of 1.72 × 106 (CV ± 0.13) and glial number of 2.25 × 106 (CV ± 0.17) in CA1, and 0.80 × 106 (CV ± 0.27) neurons and 1.05 × 106 (CV ± 0.26) glial cells in CA2–3. Comparisons with related studies show quantitative variation, but also variations in methods and applications. The results are phylogenetically consistent, apart from the neuron/glia ratio, which is remarkably higher than what is found in other species. Anat Rec, 2007. © 2007 Wiley-Liss, Inc.

The cetacean brain has long been of scientific interest, not only because of its large size – the largest in the animal kingdom – but also because of its high gyrification. It shows several adaptations to the aquatic environment, especially in the cortical arrangements of functional areas. To study structural aspects of the mysticete brain we estimated neocortical features in the common minke whale using stereological methods. The neocortex was surprisingly thick, equal to that in humans. The total neocortical neuron number was 12.8 × 109, and the total neocortical glia number 98.2 × 109. Total cell numbers in the auditory and visual cortex were also estimated, and showed that the auditory cortex contained more cells than the visual cortex. In this small sample, no sexual dimorphism was seen within the neocortex of the common minke whale. Our aim was to estimate the total cell number, cortical volume and cell density in the entire mysticete neocortex and compare the total cell number in the auditory cortex with that of the visual cortex using stereological methods. Here, we used the common minke whale as a model of all mysticetes. We wanted to compare these neocortical features to those of other mammals to forward understanding of the evolution of the mammalian brain. Anat Rec, 290:83–95, 2007. © 2006 Wiley-Liss, Inc.

The aim of this study was to quantify the total number of neurons and glial cells in the mediodorsal nucleus of the thalamus (MD) of 8 newborn human brains, in comparison to 8 adult human brains. The estimates of the cell numbers were obtained using the stereological principles of the optical fractionator. In the case of the adults, the total number of neurons in the entire MD was an average of 41% lower than in the newborn, which was statistically highly significant (P < 0.001). The estimated average total number of neurons in MD thalamus of the newborns was 11.2 million (coefficient of variation [CV] = standard deviation/mean = 0.16), compared with the adults' 6.43 million (CV = 0.15). The glial cell numbers were substantially higher in the adult brains, with an increase of almost 4 times from 10.6 million at birth to 36.3 million in the fully developed adult brain. This is the first demonstration of a higher number of human neurons in the brain of newborns compared with the adult.

Hippocampal atrophy and neuron loss are early and reproducible findings in Alzheimer's disease, and recent magnetic resonance imaging studies indicate that hippocampal atrophy may also be present in Parkinson's disease (PD). To determine whether or not cell loss occurs in PD, we estimated the total neuron and glial cell numbers as well as the total volume unilaterally in the hippocampi of eight demented PD patients and eight control subjects. Cell numbers were estimated in the neuron-containing layers of CA1, CA2-(3), CA4, the dentate gyrus, and subiculum using the optical-fractionator technique. The Cavalieri method was used to estimate the volume of the total hippocampus and its subregions. We did not find significant differences in cell numbers or volumes in PD brains when compared with control subjects. Our results thus indicate that hippocampal atrophy and cell loss are not necessarily involved in the memory impairment and dementia observed in PD. © 2006 Wiley-Liss, Inc.

OBJECTIVE: The objective of the study was to test the hypothesis that the total number of cells in the cortical part of the cerebral wall is the same in intrauterine growth-restricted (IUGR) fetuses, compared with normally grown fetuses. STUDY DESIGN: The total cell number in the cerebral wall was estimated in 9 severely affected IUGR fetuses and 15 controls using the optical fractionator. Cell numbers were estimated within 4 developmental zones. The gestational ages were 19-41 weeks. RESULTS: The total cell number in the future cortex was significantly reduced in the IUGR fetuses, compared with controls. The daily increase in brain cells in the future cortex was only half of that of the controls. In the 3 other developmental zones, no significant differences in cell numbers could be demonstrated. CONCLUSIONS: IUGR in humans is associated with a severe reduction in cortical growth and a significant decrease in cell number in the future cortex. Udgivelsesdato: 2007-Jul

Stereological cell counting was applied to post-mortem neocortices of human brains from 31 normal individuals, age 18-93 years, 18 females (average age 65 years, range 18-93) and 13 males (average age 57 years, range 19-87). The cells were differentiated in astrocytes, oligodendrocytes, microglia and neurons and counting were done in each of the four lobes. The study showed that the different subpopulations of glial cells behave differently as a function of age; the number of oligodendrocytes showed a significant 27% decrease over adult life and a strong correlation to the total number of neurons while the total astrocyte number is constant through life; finally males have a 28% higher number of neocortical glial cells and a 19% higher neocortical neuron number than females. The overall total number of neocortical neurons and glial cells was 49.3 billion in females and 65.2 billion in males, a difference of 24% with a high biological variance. These numbers can serve as reference values in quantitative studies of the human neocortex.

By means of i.c.v. administration of preaggregated oligomeric beta-amyloid (Abeta)25-35 peptide it was possible in rats to generate neuropathological signs related to those of early stages of Alzheimer's disease (AD). Abeta25-35-administration induced the deposition of endogenously produced amyloid protein. Furthermore, quantitative immunohistochemistry demonstrated time-related statistically significant increases in amyloid immunoreactivity, tau phosphorylation, microglial activation, and astrocytosis, and stereological investigations demonstrated statistically significant increased neuronal cell death and brain atrophy in response to Abeta25-35. Finally, the Abeta25-35-administration led to a reduced short-term memory as determined by the social recognition test. A synthetic peptide termed FGL derived from the neural cell adhesion molecule (NCAM) was able to prevent or, if already manifest, strongly reduce all investigated signs of Abeta25-35-induced neuropathology and cognitive impairment. The FGL peptide was recently demonstrated to be able to cross the blood-brain-barrier. Accordingly, we found that the beneficial effects of FGL were achieved not only by intracisternal, but also by intranasal and s.c. administration of the peptide. Furthermore, FGL-treatment was shown to inhibit the activity of GSK3beta, a kinase implicated in signaling regulating cell survival, tau phosphorylation and the processing of the amyloid precursor protein (APP). Thus, the peptide induced a statistically significant increase in the fraction of GSK3beta phosphorylated on the Ser9-position, a posttranslational modification known to inhibit the activity of the kinase. Hence, the mode of action of FGL with respect to the preventive and curative effects on Abeta25-35-induced neuropathological manifestations and cognitive impairment involves the modulation of intracellular signal-transduction mediated through GSK3beta.

SUMMARYThe first mathematically unbiased estimates of neocortical cell numbers are presented from the developing pig brain, including a full description of tissue processing and optimal sampling for application of the stereological optical fractionator method in this species. The postnatal development of neocortical neurons and glial cells from the experimental Göttingen minipig was compared with the postnatal development of neocortical neurons in the domestic pig. A significant postnatal development was observed in the Göttingen minipig brain for both neuronal (28%; P=0.01) and glial cells (87%; P

The total cell numbers were estimated in the neocortical part of the human telencephalon in 10 normal brains of newborn babies within four major developmental zones: the cortical plate/marginal zone, the subplate, the intermediate zone and the ventricular/subventricular zone. Furthermore, the total number of neuron and glial cells was estimated in the cortical plate. The gestational ages ranged from 38 + 0-42 + 5 weeks + days of gestation. The mean total cell number was 32.6 x 10(9) (coefficient of error = 0.04) and the total number of neurons in the cortical plate 19.8 x 10(9) (coefficient of error = 0.06). This indicates that the total number of neocortical neurons equals the total number in the adults, which, however, is not the case for the glial cells.