A novel workflow for unbiased 3D quantification of autophagosomes in Arabidopsis thaliana roots.

Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP-ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z-direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple AtATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP-AtATG8e marker, that most probably does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume positively correlated with the intensity of autophagy induction. Compared with the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP-ATG8 markers.

Improving Cavalieri volume estimation based on non-equidistant planar sections: The trapezoidal estimator.

The Cavalieri estimator allows one to infer the volume of an object from area measurements in equidistant planar sections. It is known that applying this estimator in the non-equidistant case may inflate the coefficient of error considerably. We therefore consider a newly introduced variant, the trapezoidal estimator, and make it available to practitioners. Its typical variance behaviour for natural objects is comparable to the equidistant case. We state this unbiased estimator, describe variance estimates and explain how the latter can be simplified under rather general but realistic models for the gaps between sections. Simulations and an application to a synthetic area function based on parietal lobes of 18 monkeys illustrate the new methods.

Neurodegeneration and Astrogliosis in the Human CA1 Hippocampal Subfield Are Related to hsp90ab1 and bag3 in Alzheimer's Disease.

Alzheimer's disease (AD), the most prevalent neurodegenerative disorder, is characterized by executive dysfunction and memory impairment mediated by the accumulation of extracellular amyloid-ß peptide (Aß) and intracellular hyperphosphorylated tau protein. The hippocampus (HIPP) is essential for memory formation and is involved in early stages of disease. In fact, hippocampal atrophy is used as an early biomarker of neuronal injury and to evaluate disease progression. It is not yet well-understood whether changes in hippocampal volume are due to neuronal or glial loss. The aim of the study was to assess hippocampal atrophy and/or gliosis using unbiased stereological quantification and to obtain hippocampal proteomic profiles related to neurodegeneration and gliosis. Hippocampal volume measurement, stereological quantification of NeuN-, Iba-1- and GFAP-positive cells, and sequential window acquisition of all theoretical mass spectrometry (SWATH-MS) analysis were performed in AD and non-AD cases. Reduced hippocampal volume was identified using the Cavalieri probe, particularly in the CA1 region, where it correlated with neuronal loss and astrogliosis. A total of 102 downregulated and 47 upregulated proteins were identified in the SWATH-MS analysis after restrictive filtering based on an FC > 1.5 and p value < 0.01. The Hsp90 family of chaperones, particularly BAG3 and HSP90AB1, are closely related to astrocytes, indicating a possible role in degrading Aß and tau through chaperone-mediated autophagy.

The importance of volume and area fractions of cerebellar volume and vermian subregion areas: a stereological study on MR images.

PURPOSE: Age, gender, and body size are important factors which are affecting the cerebellar volume (CV). Many neurological diseases lead changes in CV. The aim of this study is to measure CV and the total intracranial volume (TIV) for both genders on magnetic resonance images (MRI), to calculate the CV/TIV volume fraction, and also to determine the normal values that can be regarded clinically significant by determining the total vermis area and vermian subregion areas (V1, V2, and V3). METHODS: In this retrospective study, MR images (without any pathological findings) of 200 individuals (100 female, 100 male) between the ages of 20-40 were used. CV and CV/TIV volume fractions, vermian subregion areas, and area fractions were calculated by using the Stereoinvestigator 8.0 (Microbrightfield, USA) software. The volumetric calculations were performed by the point counting method according to the Cavalieri principle, which is one of the volume calculation methods in stereology. Total CV, TIV, cerebellar vermis areas (V1, V2, and V3), and total cerebellum area were measured separately for both groups. RESULTS: The volume of cerebellum was 120.53 ± 11.1 cm3 in males, 105.99 ± 11.2 cm3 in females, TIV was 1304.99 ± 91.7 cm3 in males and 1155.15 ± 85.7 cm3 in females. CV and TIV were statistically higher in males (p = 0.001, p = 0.001 respectively). It was observed that the differences between the genders in terms of CV/TIV disappeared (p = 0.679). The total vermis area was 11.59 ± 1.3 cm2 in males and 10.85 ± 1.3 cm2 in females. V1 area, V3 area, and the total vermis area were found statistically higher in males (p = 0.05, p = 0.006, p = 0.007 respectively). It was determined that the area fraction of V2 was higher in females when the fractions of V1, V2, and V3 to the total vermis area were examined (p = 0.03). CONCLUSION: We believe that the normal values of CV, TIV, and vermian subregion areas, determined by stereological method, will contribute to the diagnosis and the treatment plan of the clinical pathological evaluations in adults and children.

Quantitative changes in testicular structure and function in rat exposed to mobile phone radiation.

The possible effects of the electromagnetic fields (EMF) generated by mobile phones on reproductive functions have been discussed in recent years. The aim of this study was to evaluate the effects of EMF emitted from mobile phones on the rat testis morphology and histopathology using stereological techniques. We also investigated cortisol, testosterone, FSH and LH levels. A total of thirty-two (n = 32) male Wistar albino rats were used in this study. Animals were randomly divided into four groups as control (C, n = 8), sham (Sh, n = 8), mobile phone speech (Sp, n = 8) and mobile phone standby (ST by). Morphometric measurements were made with the help of a computer-assisted stereological analysis system. The testis weight and volume were significantly lower in the EMF exposed groups. The mean volume fraction of interstitial tissue was higher, but the volume fraction of tubular tissue was lower in the EMF-exposed groups. The mean tubular and germinal tissue volume, seminiferous tubule diameter and germinal epithelium height were also lower in EMF exposed groups. The cortisol levels in the EMF-exposed groups were significantly higher. In conclusion, the EMF created by mobile phones caused morphologic and histological changes by the affecting germinal epithelium tissue negatively.

Effects of platelet-rich plasma on cartilage regeneration after costal cartilage resection: a stereological and histopathological study.

BACKGROUND: In cases of congenital chest wall deformities, it is important to maintain the flexibility of the chest wall after rib cartilage resection. In this study, we aimed to determine the regeneration capability of cartilage and the effects of platelet-rich plasma (PRP) on the regeneration process. METHODS: A total of 16 four-week-old New Zealand rabbits were used in this study. In the 4th-5th right costal cartilages, the perichondrial sheaths were dissected and costal cartilages were excised. Then, the perichondrial sheaths were closed with absorbable material in the sham group (n = 8), and this was done after replacing PRP in the PRP group (n = 8). The left costal cartilages of the animals were used as controls. The volumes of the costal cartilages and their perichondrial sheaths were estimated using Cavalieri's principle. In addition, the mean numerical densities of the chondroblasts and chondrocytes per square millimetre were estimated using unbiased counting frames. RESULTS: In the PRP and sham groups, the volumes of the cartilages and perichondrial sheaths were higher than those of the control group (p < 0.05). The numerical densities of the chondroblasts and chondrocytes increased more in the PRP group than in the sham group (p < 0.05). CONCLUSIONS: Applying PRP after resection may provide better healing and faster regeneration of cartilage.

Absolute number of parvicellular and magnocellular neurons in the red nucleus of the rat midbrain: a stereological study.

The absolute number of parvicellular and magnocellular neurons in the red nucleus was estimated using design-based stereological counting methods and systematic random sampling techniques. Six young adult male rats, and a complete set of serial 40-µm glycolmethacrylate sections for each rat, were used to quantify neuronal numbers. After a random start, a systematic subset (i.e. every third) of the serial sections was used to estimate the total volume of the red nucleus using Cavalieri's method. The same set of sampled sections was used to estimate the number of neurons in a known subvolume (i.e. the numerical density Nv ) by the optical disector method. Multiplication of the total volume by Nv yielded the absolute number of neurons. It was found that the right red nucleus consisted, on average, of 8400 parvicellular neurons (with a coefficient of variation of 0.16) and 7000 magnocellular neurons (0.12). These total neuronal numbers provide important data for the transfer of information through these nuclei and for species comparisons.

Delayed post-treatment with bone marrow-derived mesenchymal stem cells is neurorestorative of striatal medium-spiny projection neurons and improves motor function after neonatal rat hypoxia-ischemia.

Perinatal hypoxia-ischemia is a major cause of striatal injury and may lead to cerebral palsy. This study investigated whether delayed administration of bone marrow-derived mesenchymal stem cells (MSCs), at one week after neonatal rat hypoxia-ischemia, was neurorestorative of striatal medium-spiny projection neurons and improved motor function. The effect of a subcutaneous injection of a high-dose, or a low-dose, of MSCs was investigated in stereological studies. Postnatal day (PN) 7 pups were subjected to hypoxia-ischemia. At PN14, pups received treatment with either MSCs or diluent. A subset of high-dose pups, and their diluent control pups, were also injected intraperitoneally with bromodeoxyuridine (BrdU), every 24h, on PN15, PN16 and PN17. This permitted tracking of the migration and survival of neuroblasts originating from the subventricular zone into the adjacent injured striatum. Pups were euthanized on PN21 and the absolute number of striatal medium-spiny projection neurons was measured after immunostaining for DARPP-32 (dopamine- and cAMP-regulated phosphoprotein-32), double immunostaining for BrdU and DARPP-32, and after cresyl violet staining alone. The absolute number of striatal immunostained calretinin interneurons was also measured. There was a statistically significant increase in the absolute number of DARPP-32-positive, BrdU/DARPP-32-positive, and cresyl violet-stained striatal medium-spiny projection neurons, and fewer striatal calretinin interneurons, in the high-dose mesenchymal stem cell (MSC) group compared to their diluent counterparts. A high-dose of MSCs restored the absolute number of these neurons to normal uninjured levels, when compared with previous stereological data on the absolute number of cresyl violet-stained striatal medium-spiny projection neurons in the normal uninjured brain. For the low-dose experiment, in which cresyl violet-stained striatal medium-spiny neurons alone were measured, there was a lower statistically significant increase in their absolute number in the MSC group compared to their diluent controls. Investigation of behavior in another cohort of animals showed that delayed administration of a high-dose of bone marrow-derived MSCs, at one week after neonatal rat hypoxia-ischemia, improved motor function on the cylinder test. Thus, delayed therapy with a high- or low-dose of adult MSCs, at one week after injury, is effective in restoring the loss of striatal medium-spiny projection neurons after neonatal rat hypoxia-ischemia and a high-dose of MSCs improved motor function.

Sampling theory and automated simulations for vertical sections, applied to human brain.

In recent years, there have been substantial developments in both magnetic resonance imaging techniques and automatic image analysis software. The purpose of this paper is to develop stereological image sampling theory (i.e. unbiased sampling rules) that can be used by image analysts for estimating geometric quantities such as surface area and volume, and to illustrate its implementation. The methods will ideally be applied automatically on segmented, properly sampled 2D images - although convenient manual application is always an option - and they are of wide applicability in many disciplines. In particular, the vertical sections design to estimate surface area is described in detail and applied to estimate the area of the pial surface and of the boundary between cortex and underlying white matter (i.e. subcortical surface area). For completeness, cortical volume and mean cortical thickness are also estimated. The aforementioned surfaces were triangulated in 3D with the aid of FreeSurfer software, which provided accurate surface area measures that served as gold standards. Furthermore, a software was developed to produce digitized trace curves of the triangulated target surfaces automatically from virtual sections. From such traces, a new method (called the 'lambda method') is presented to estimate surface area automatically. In addition, with the new software, intersections could be counted automatically between the relevant surface traces and a cycloid test grid for the classical design. This capability, together with the aforementioned gold standard, enabled us to thoroughly check the performance and the variability of the different estimators by Monte Carlo simulations for studying the human brain. In particular, new methods are offered to split the total error variance into the orientations, sectioning and cycloid components. The latter prediction was hitherto unavailable--one is proposed here and checked by way of simulations on a given set of digitized vertical sections with automatically superimposed cycloid grids of three different sizes. Concrete and detailed recommendations are given to implement the methods.

Applying the area fraction fractionator (AFF) probe for total volume estimations of somatic, dendritic and axonal domains of the nigrostriatal dopaminergic system in a murine model.

BACKGROUND: The Cavalieri estimator is used for volume measurement of brain and brain regions. Derived from this estimator is the Area Fraction Fractionator (AFF), used for efficient area and number estimations of small 2D elements, such as axons in cross-sectioned nerves. However, to our knowledge, the AFF has not been combined with serial sectioning analysis to measure the volume of small-size nervous structures. NEW METHOD: Using the nigrostriatal dopaminergic system as an illustrative case, we describe a protocol based on Cavalieri's principle and AFF to estimate the volume of its somatic, nuclear, dendritic, axonal and axon terminal cellular compartments in the adult mouse. The protocol consists of (1) systematic random sampling of sites within and across sections in regions of interest (substantia nigra, the nigrostriatal tract, caudate-putamen), (2) confocal image acquisition of sites, (3) marking of cellular domains using Cavalieri's 2D point-counting grids, and 4) determination of compartments' total volume using the estimated area of each compartment, and between-sections distance. RESULTS: The volume of the nigrostriatal system per hemisphere is ∼0.38 mm3, with ∼5 % corresponding to perikarya and cell nuclei, ∼10 % to neuropil/dendrites, and ∼85 % to axons and varicosities. COMPARISON WITH EXISTING METHODS: In contrast to other methods to measure volume of discrete objects, such as the optical nucleator or 3D reconstructions, it stands out for its versatility and ease of use. CONCLUSIONS: The use of a simple quantitative, unbiased approach to assess the global state of a system may allow quantification of compartment-specific changes that may accompany neurodegenerative processes.

Calculation of cerebral hemispheres volume values (grey matter, white matter and lateral ventricle) of sheep and goat: A stereological study.

Stereology is a discipline that allows us to obtain quantitative information about the geometric structure of three-dimensional objects. In this study, the volume of grey matter (GM), white matter (WM), and lateral ventricle (LV) of the cerebral hemispheres (CH) in sheep and goats were calculated. For this purpose, six healthy male sheep and goat brains (1-2 years old) without any anomaly were used. Brains were fixed with 10% formaldehyde in the skull. The skull was opened using standard anatomical dissection methods, and the brains were carefully removed. Brain weight and volume were measured (using Archimedes' principle) after the meninges were removed. The cerebral hemispheres were separated from the other parts of the brain by a section made in front of the rostral colliculus. In the same way, the weight and volume of the cerebral hemispheres were measured. Afterward, the cerebral hemispheres were blocked with agar, and transversal cross sections (from rostral to caudal) with an average thickness of 3.42 mm were taken from the cerebral hemispheres. Grey matter was stained with Berlin blue macroscopic staining method. The stained cross sections were scanned at 600 dpi resolution, and a point counting grid was placed on the images with the ImageJ software. Cavalieri's principle calculated the surface area and volume measurements of the grey matter, white matter, and lateral ventricle. GM, WM, and LV volumes in sheep and goat cerebral hemispheres were calculated as 54.94, 21.48 and 3.06 mL in sheep, 57.46, 24.13 and 3.12 mL in goats, respectively. The percentages of these structures in the total hemisphere volume were 71.83%, 28.17% and 4.00% in sheep, 70.42%, 29.58% and 3.82% in goats, respectively. Asymmetry was not observed in cerebral hemispheres in both species. A difference was found in the WM, LV and LV: CH ratios in the right/left comparison of the goat (p < 0.05). In comparing sheep and goats, a significant difference was observed in WM right, WM left, WM total, CH left and CH total (p < 0.05). In conclusion, the cerebral hemispheres' grey matter and white matter ratio are frequently used to diagnose neurodegenerative diseases. In recent years, the increase in neurodegenerative disease models in farm animals has been enormous. It is thought that these values obtained from healthy animals in the current study will be important for such experimental studies in the future.

Application of design-based stereology for estimation of absolute volume and surface area of the articular and calcified cartilage compartments of undecalcified human femoral heads.

Design-based stereological methods using systematic uniform random sampling, the Cavalieri estimator and vertical sections are used to investigate undecalcified human femoral heads. Ten entire human femoral heads, obtained from normal women and normal men, were systematically sampled and thin undecalcified vertical sections were obtained. Absolute volumes and surface areas of the entire femoral head, the articular cartilage and the calcified cartilage compartments were estimated. In addition, the average thickness of the articular cartilage and the calcified cartilage were calculated. The stereological procedures applied to the human femoral heads resulted in average coefficient of errors, which were 0.03-0.06 for the volume estimates and 0.03-0.04 for the surface area estimates. We conclude that design-based stereology using the Cavalieri estimator and vertical sections can successfully be used in large undecalcified tissue specimens, like the human femoral head, to estimate the absolute volume and surface area of macroscopic as well as of microscopic tissue compartments. The application of well-known design-based stereological methods carries potential advantage for investigating the pathology in inflammatory and degenerative joint diseases.

Brain Structural Changes in Schizophrenia Patients Compared to the Control: An MRI-based Cavalieri's Method.

Introduction: Schizophrenia is a severe psychotic brain disorder. One of the potential mechanisms underlying this disease may be volumetric changes in some brain regions. The present study aimed to employ magnetic resonance imaging (MRI) to estimate and quantitatively analyze the brain of patients with schizophrenia compared to the controls. Methods: This case-control study was conducted on MRI scans of 20 patients with schizophrenia and 20 healthy controls in Zahedan City, Southeastern Iran. MRIs with 4 mm slice thickness and 5 mm intervals in coronal and sagittal planes were captured. Then, quantitative parameters, including volume and volume density of various brain regions, were estimated in both groups using Cavalieri's point counting method. Data analyses were performed using the Mann-Whitney U test. Results: The findings of this investigation revealed that volumes of gray matter, hippocampus, and gray/white matter in patients with schizophrenia were significantly lower than the controls (P<0.05). The volumes of lateral ventricles in patients with schizophrenia (36.60±4.32 mm3) were significantly higher than the healthy individuals (30.10±7.98 mm3). However, there were no statistically significant differences between the two groups regarding the changes in the brain's total volume, cerebral hemispheres, white matter, brain stem, cerebellum, and corpus callosum (P>0.05). Conclusion: Volumetric estimations on brain MRI-based stereological technique can be helpful for elucidation of structural changes, following up the treatment trends, and evaluating the therapeutic situations in schizophrenia patients. Volumetric alternations in specific brain areas might be linked to cognitive impairments and the severity of symptoms in patients with schizophrenia. Further research is needed in this regard. Highlights: Volumetric changes occur in certain regions of the brain of schizophrenia patients.Structural changes in the brain of schizophrenia patients are associated with the severity of clinical manifestations.A brain MRI-based stereological technique can clarify neuropathology and assess therapeutic efficiency in patients with schizophrenia. Plain Language Summary: Schizophrenia is a severe neuropsychiatric disorder with worldwide prevalence that disrupts a person's social life. It's characterized by progressive neuroanatomical alterations in both gray and white matter in different brain regions and associated with changes in the structural and functioning of some critical brain circuits. Several factors have been suggested to be involved in the development and progression of the disease including alternations and disconnection in myelin, genetic factors, neurodegenerative process, neuroinflammation, neurodevelopmental deficiencies, the number of dopaminergic neurons and volumetric changes in different areas of the brain. It has shown that quantitative volumetric brain measurements on magnetic resonance imaging (MRI) scans in patients with neurodegenerative disease owing to selective regional atrophy are beneficial for clinicians to ascertain disease progression and to evaluate volume alternations and response to treatment. Thus, we investigated structural changes of the brain in schizophrenia patients on MR images using accurate Cavalieri's estimation and compared to healthy controls. The findings demonstrated that some structural changes occurs in various brain areas which involved in many critical roles in normal brain's functionality and connectivity. On the other hand, these changes are associated with cognitive impairments and the severity of clinical symptoms in patients with schizophrenia. It's appears that elucidation of the different pathways of various structural abnormalities related to schizophrenia is required to recognize and determine the role of discrete pathophysiological phenomena in mental illness development and progress.

Applications of various stereological tools for estimation of biological tissues.

To provide concise and brief important stereological application methods and techniques for estimating biological tissues. Stereology studies the quantity of biological tissue using little practice and the low price of counting and preparing tissue slices to obtain direct and accurate results. Since their establishment, the stereological techniques underwent much improvement, thus allowing more precise analysis of target structures using various approaches. Using stereological tools, advances in stereological techniques made the target tissues or organs represented by 2D instead of 3D dimensions. Process tools estimate volume, area and length. According to the exciting tissue and aims, the stereological tools perform differently. This review summarizes various stereological tools and techniques, providing brief information about the orientation method, slicer probe method, Delesse's principle, Cavalieri principle, disector, fractionator, nucleator, virtual cycloids and saucer, which are described in detail.

Comparative evaluation of glomerular morphometric techniques reveals differential technical artifacts between focal segmental glomerulosclerosis and normal glomeruli.

Morphometric estimates of mean or individual glomerular volume (MGV, IGV) have biological implications, over and above qualitative histologic data. However, morphometry is time-consuming and requires expertise limiting its utility in clinical cases. We evaluated MGV and IGV using plastic- and paraffin-embedded tissue from 10 control and 10 focal segmental glomerulosclerosis (FSGS) mice (aging and 5/6th nephrectomy models) using the gold standard Cavalieri (Cav) method versus the 2-profile and Weibel-Gomez (WG) methods and a novel 3-profile method. We compared accuracy, bias and precision, and quantified results obtained when sampling differing numbers of glomeruli. In both FSGS and controls, we identified an acceptable precision for MGV of 10-glomerular sampling versus 20-glomerular sampling using the Cav method, while 5-glomerular sampling was less precise. In plastic tissue, 2- or 3-profile MGVs showed greater concordance with MGV when using Cav, versus MGV with WG. IGV comparisons using the same glomeruli reported a consistent underestimation bias with both 2- or 3-profile methods versus the Cav method. FSGS glomeruli showed wider variations in bias estimation than controls. Our 3-profile method offered incremental benefit to the 2-profile method in both IGV and MGV estimation (improved correlation coefficient, Lin's concordance and reduced bias). In our control animals, we quantified a shrinkage artifact of 52% from tissue processed for paraffin-embedded versus plastic-embedded tissue. FSGS glomeruli showed overall reduced shrinkage albeit with variable artifact signifying periglomerular/glomerular fibrosis. A novel 3-profile method offers slightly improved concordance with reduced bias versus 2-profile. Our findings have implications for future studies using glomerular morphometry.

A study on the correlation between spleen volume estimated via cavalieri principle on computed tomography images with basic hemogram and biochemical blood parameters.

Considering its hematological and immunological functions, spleen is a very important organ. A change occurs in its size as the spleen performs these functions. This study aims to examine the possible relationships between spleen volume and the basic hemogram and biochemical parameters in serum. Multidetector computed tomography images and basic hemogram and biochemical parameters of 74 adult individuals, 34 male and 40 female, who were found to be healthy, were used in the study. Spleen volume was estimated using the Cavalieri method on multidetector computed tomography images and the correlations between the volume value with basic hemogram and biochemistry parameters were researched. While negative significant correlations were found between the estimated spleen volume and lymphocyte percentage (r=-0.224) and platelet level (r=-0.271); positive significant correlations were found between hemoglobin level (r=0.228), hematocrit level (r=0.237), alanine aminotransferase (r=0.345), and erythrocyte level (r=0.375). As a result of this study, a relationship was found between spleen volume and lymphocyte percentage, hematocrit level, erythrocyte level, platelet level, and alanine aminotransferase level in serum. We believe that the results of the study will provide a larger perspective to clinicians in the diagnosis of diseases associated with spleen.

Estimation of Spleen Volume Using MRI Segmentation: Would One Slice Be Enough?

This study aimed to establish spleen volume in adult Sudanese using manual segmentation and the Cavalieri method with magnetic resonance images (MRI). A total of 345 abdominal MRI scans for adults were retrospectively studied for patients who underwent abdomen MRI between September to October 2016. The data were collected from two scanning centers in Khartoum, Sudan. For each series of sectional images, the spleen was manually segmented, and the volume was calculated using matrix laboratory (MATLAB) code. Furthermore, abdominal circumferences and diameters, in addition to L1 body dimension and spinal canal, were measured.  The mean splenic volume in our study was 187.2 cm3, without a significant change with age (P = 0.269). The average volume in males was 223.5 cm3 while in females it was 170.27 cm3. The average coefficient error (CE) was 0.029. A positive significant correlation was found between the volume and spleen length (beta standardized coefficient = 0.781, P < 0.05). The mean length was 9.18 cm. Additionally, our data showed an insignificant correlation between the volume and the other measurements, except for the abdomen transverse diameter (beta standardized coefficient = 0.267, P < 0.05).  The results of our study coincided with previous studies in the normal range of spleen volume and the strong correlation with length, regardless of the irregular shape of the spleen. In addition, manual segmentation was a reliable method to measure spleen volume objectively. However, our single-slice measurements were insufficient.

The Effect of Short-Term Exposure to Cadmium on the Expression of Vascular Endothelial Barrier Antigen in the Developing Rat Forebrain and Cerebellum: A Computerized Quantitative Immunofluorescent Study.

Clinical and laboratory studies have shown that environmental exposure to cadmium produces damage to several organs, including bones, lungs, and kidneys. The involvement of cadmium in central nervous system (CNS) disorders has also been widely reported, but the precise pathophysiological mechanism is not yet fully understood. Children who were exposed to cadmium during pregnancy are known to suffer from developmental delays, learning difficulties, attention deficit hyperactivity disorder (ADHD), and other cognitive and neurobehavioral deficits. Results from numerous studies suggest that dysfunction of the blood-brain barrier (BBB) structures is an important step in the neurotoxicity of cadmium. A rat-specific BBB marker protein, the endothelial barrier antigen (EBA), has been previously isolated and classified by Sternberger and others. The mouse IgG1 clone, anti-endothelial barrier antigen (anti-EBA), detects a protein triplet (23.5kDa, 25 kDa, and 30kDa) localized to the luminal surface of central and peripheral nervous system (CNS and PNS) vascular endothelial cells with selective permeability barrier functions. This marker has been widely used for characterizing BBB alterations under demyelinating, inflammatory, and other CNS pathologies. Many studies have been published using the rat model system for studying the neurotoxic effect of acute and chronic exposure to cadmium. We applied the indirect immunofluorescent techniques using the anti-EBA antibody in conjunction with the Olympus cellSens computerized image analysis to detect and quantify the surface areas of BBB-competent microvessel profiles in paraformaldehyde-fixed, paraffin-embedded brains of term-delivered young rats after intraperitoneal injection of a single dose of cadmium chloride. We detected a statistically significant reduction in EBA-positive microvessel surface areas in the forebrain (t = 5.86, df = 1789, p-value < 0.001) and cerebellum (t=73.40, df=1337, p < 0.001) of cadmium-treated rats compared to the normal controls. Thus, this study supports the hypothesis that the EBA is a sensitive and measurable indicator for quantitative assessment of the impact of cadmium exposure in the developing rat brain.

Improving the measurement of intrinsic foot muscle morphology and composition from high-field (7T) magnetic resonance imaging.

Magnetic resonance imaging (MRI) can be used to quantify intrinsic foot muscle morphology and composition. Due to the high spatial resolution required to adequately capture the architecturally complex anatomy, manual segmentation is time consuming and not clinically feasible. The aim of this study was to evaluate if a reduced number of MRI slices can be used to accurately estimate intrinsic foot muscle volume and composition. A three-dimensional 2-point Dixon sequence of the whole foot was acquired at 7-Tesla for thirteen asymptomatic individuals and twenty individuals with plantar heel pain. Slice intervals of 2, 3, 5, 10, 15 and 30 were used to calculate alternative muscle volume and composition, and were compared to reference values calculated from every available slice. Agreement between methods was assessed by calculating mean differences and 95% limits of agreement, and inspection of Bland -Altman plots. In both groups, slice intervals of 2, 3 and 5 provided excellent precision for all muscles (measurement error < 1%). Larger slice intervals of 10, 15 and 30 provided excellent precision for some muscles, but for other muscles (e.g. small forefoot muscles), error was up to 7.3%. Bland-Altman plots showed no systematic measurement bias. This study provides a quantitative basis for selecting a reduced number of slices to measure intrinsic foot muscle volume and composition from MRI. A slice interval of 10 may provide a balance between efficiency (36 mins vs. 6 h) and accuracy (error < 2.4%) across all intrinsic foot muscles in asymptomatic individuals and those with plantar heel pain.

Comparison of volume of the forebrain, subarachnoid space and lateral ventricles between dogs with idiopathic epilepsy and controls using a stereological approach: Cavalieri's principle.

BACKGROUND: Canine idiopathic epilepsy (IE) is the most common chronic neurological brain disease in dogs, yet it can only be diagnosed by exclusion of all other potential causes. In people, epilepsy has been associated with a reduction in brain volume. The objective was to estimate the volume of the forebrain (FB), subarachnoid space (SAS) and lateral ventricles (LV) in dogs with IE compared to controls using Cavalieri's principle. MRI scans of case and control dogs were identified from two neurology referral hospital databases. Eight breeds with increased odds of having IE were included: Golden Retriever, Labrador Retriever, Cocker Spaniel, Border terrier, German Shepherd dog, Parson Jack Russell terrier, Boxer, and Border Collie. Five dogs of each breed with IE and up to five controls were systematically and uniformly randomly sampled (SURS). The volume of the FB, SAS and LV were estimated from MRI scans by one blinded observer using Cavalieri's principle. RESULTS: One hundred-two dogs were identified; 56 were diagnosed with IE and 46 were controls. There was no statistically significant difference in FB, SAS and LV volume between dogs with IE and controls. Dogs with a history of status epilepticus had significantly larger FB than those without (p = 0.05). There was a border-line trend for LV volume to increase with increasing length of seizure history in the IE group (p = 0.055). CONCLUSION: The volumes of the FB, SAS and LV are not different between dogs with IE and controls, so IE remains a diagnosis of exclusion with no specific neuroanatomical biomarkers identified. This is the first time FB and SAS volume has been compared in dogs with IE. Unfortunately, we have shown that the results reporting significantly larger FBs in dogs with status epilepticus and LV volume increase with length of seizure history were likely confounded by breed and should be interpreted cautiously. Whilst these associations are interesting and clinically relevant, further investigation with breed-specific or larger, breed-diverse populations are required to permit strong conclusions. The Cavalieri principle provided an effective estimation of FB, SAS and LV volumes on MRI, but may be too time-intensive for use in clinical practice.