Stereology in Materials Science

Quantitative analysis of populations having ageometric structure,which has developed into aspecial scientific subject called microstructology orstereology,is of great importance to thecharacterization and evaluation of microstructuresand their evolution in various processes.This paper,besides a brief discussion on those topics such as therecent developments of computer assisted imageanalysis,mathematical morphology,and fractalanalysis,will mainly focus on the scope,fundamen-tals,present status,and perspectives of classicalstereology.Several case examples of its applicationto materials science will also be given.

Protective effect of co-enzyme Q10 on testicular tissue and sperm parameters in adult male rats treated with Sunset Yellow FCF

Objective:To determine the protective effect of co-enzyme Q10(CoQ10)on testicular tissue and sperm parameters in male rats treated with SunsetYellow FCF.Methods:Sixty male Sprague-Dawley rats were randomly divided into 6 groups of the control,CoQ10(10 mg/kg/day),low dose of Sunset Yellow(2.5 mg/kg),high dose of Sunset Yellow(70 mg/kg),low dose of Sunset Yellow(2.5 mg/kg)plus CoQ10,and high dose of Sunset Yellow(70 mg/kg)plus CoQ10.The drugs were administered via daily oral gavages for 6 weeks.At the end of the experiment,sperm analysis,stereological and histological assessments of the testis were carried out.Results:The normal morphology(by 41.1%)and progressive spermatozoa(by 74.8%),testicle volume(by 33.4%),lumen volume(by 38.3%),interstitial tissue volume(by 44.7%),seminiferous tubule volume(by 40.7%),and number of spermatogonia(by 53.9%)and Leydig cells(by 70.7%)reduced in the rats that received high doses of Sunset Yellow in comparison to the control group.Nonetheless,all these alterations were recovered by CoQ10 treatment in the CoQ10 plus high dose of Sunset Yellow group.Furthermore,low doses of Sunset Yellow did not affect different parameters of the testis and sperm.Conclusions:CoQ10 could,to some extent,prevent structural changes of the testis induced by the high dose of SunsetYellow.

Evaluation of stereology for snow microstructure measurement and microwave emission modeling:a case study

Reliablemicrostructuremeasurement of snow is a requirement for microwave radiative transfer model validation.Snow specific surface area(SSA)can be measured using stereological methods,in which snow samples are cast in the field and photographed in the laboratory.Processing stereology photographs manually by counting intersections of test cycloids with air-ice boundaries reduces the problems in binary segmentation.This paper is a case study to evaluate the repeatability of the manually stereology interpretation by two independent research groups.We further assessed how uncertainty in snow SSA influences simulated brightness temperature(TB)driven by the Microwave Emission Model of Layered Snowpacks(MEMLS),and how stereology compares to Near Infrared(NIR)camera and hand lens.Data was obtained from two alpine snow profiles from Steamboat Springs,Colorado.Results showed that stereological SSA values measured by two groups are highly consistent,and the ground radiometer measured T_(B)at 19 and 37 GHz was successfully predicted(RMSE<3.8 K);simulations using NIR SSA and hand-lens geometric grain size(Dg)measurements have larger errors.This conclusion was not sensitive to uncertainty in the free parameters of TB modeling.

Effect of benzene on the cerebellar structure and behavioral characteristics in rats

Objective: To investigate the effects of benzene on rat's cerebellum structure and behavioral characteristics, including anxiety and motor impairment.Methods: Twenty rats were randomly allocated into two groups orally receiving distilled water and benzene(200 mg/kg/day). A total of 10 rats were used at the beginning of benzene exposure. Two rats died during benzene treatment and 8 rats remained for evaluation of the behavioral test and finally 6 rats underwent histological assessment. At the end of the 4th week, motor function and anxiety were evaluated in rotarod test and elevated plus maze, respectively. Besides, the cerebellum was dissected for structural assessment using stereological methods.Results: Performance of the benzene-treated rats in fixed and accelerating speed rotarod was impaired and their riding time(endurance) was lower compared to the control group(P = 0.02). The benzene-treated rats also spent less time in the open arms and had fewer entrances to the open arms in comparison to the control group, indicating anxiety(P = 0.01). The total volume of the cerebellar hemisphere, its cortex, intracerebellar nuclei, total number of the Purkinje, Bergmann, Golgi, granule, neurons and glial cells of the molecular layer, and neurons and glial cells of the intracerebellar nuclei were reduced by 34%-76% in the benzene-treated rats in comparison to the distilled water group(P = 0.003). The most cell loss was seen in Bergmann glia. Conclusions: The structure of cerebellum altered after benzene treatment. In addition, motor impairment and anxiety could be seen in benzene-treated rats.

Estimation of the density of neural,glial,and endothelial lineage cells in the adult mouse dentate gyrus

The dentate gyrus subregion of the mammalian hippocampus is an adult neural stem cell niche and site of lifelong neurogenesis.Hypotheses regarding the role of adult-born neuron synaptic integration in hippocampal circuit function are framed by robust estimations of adultborn versus pre/perinatally-born neuron number.In contrast,the non-neurogenic functions of adult neural stem cells and their immediate progeny,such as secretion of bioactive growth factors and expression of extracellular matrix-modifying proteins,lack similar framing due to few estimates of their number versus other prominent secretory cells.Here,we apply immunohistochemical methods to estimate cell density of neural stem/progenitor cells versus other major classes of glial and endothelial cell types that are potentially secretory in the dentate gyrus of adult mice.Of the cell types quantified,we found that GFAP^(+)SOX2^(+)stellate astrocytes were the most numerous,followed by CD31^(+)endothelia,GFAP-SOX2^(+)intermediate progenitors,Olig2^(+)oligodendrocytes,Iba1+microglia,and GFAP^(+)SOX2^(+)radial glia-like neural stem cells.We did not observe any significant sex differences in density of any cell population.Notably,neural stem/progenitor cells were present at a similar density as several cell types known to have potent functional roles via their secretome.These findings may be useful for refining hypotheses regarding the contributions of these cell types to regulating hippocampal function and their potential therapeutic uses.All experimental protocols were approved by the Ohio State University Institutional Animal Care and Use Committee(protocol#2016A00000068)on July 14,2016.

Anti-inflammatory, anti-oxidative and anti-apoptotic effects of Heracleum persicum L. extract on rats with gentamicin-induced nephrotoxicity

Objective:To evaluate the effect of Heracleum persicum L.against gentamicin-induced nephrotoxicity in rats.Methods:Thirty-six Wistar rats were divided into 6 groups including control(normal saline),gentamicin(80 mg/kg/d for 10 d),Heracleum persicum(750 mg/kg/d),and gentamicin(10 d)+Heracleum persicum extract at three different doses(250,500,and 750 mg/kg/d for 40 d).Urine creatinine,urea,protein,and albumin levels were determined.In addition,serum urea,creatinine,sodium,potassium,cytokines(TNF-α,IL-1β,IL-6,and IL-10),glutathione peroxidase activity,total antioxidant capacity,kidney malondialdehyde,stereological parameters,and expressions of apoptosis-related genes(p53,Bax,Bcl-2,and caspase-3)were measured.The LD50 of Heracleum persicum extract was determined based on Lorke’s method.Histopathological evaluation was also performed.Results:In addition to decreased urine protein and albumin,and increased creatinine and urea,co-treatment with gentamicin and Heracleum persicum significantly reduced levels of creatinine and urea,and increased sodium and potassium in serum.Heracleum persicum treatment also improved stereological parameters and serum inflammatory cytokines.There was a significant increase in serum glutathione peroxidase activity and total antioxidant capacity as well as a reduction in malondialdehyde level.Furthermore,treatment with Heracleum persicum extracts downregulated p53,caspase-3,and Bax and upregulated Bcl-2 expressions.In histopathological evaluation,Heracleum persicum extracts showed protection against gentamicin-induced renal damages.Conclusions:Heracleum persicum exhibits protective effects against gentamicin-induced structural and functional renal impairments.

GEOMETRIC MODEL AND APPLIED METHOD FOR THE CALCULATION OF GRAIN SIZE DISTRIBUTIONS

Most of the available methods for the calculation of the three dimensional(3D)grain size dis-tribution functions(SDF)are based on the S model and lead a grave systematical error.Theorigin is the basic supposition of spherical grains in the S model,which does not correspondwith the feature of real grains.A new model called A model is developed based on the hypothe-sis of polvhedral grains.The probability functions of the A model and the method to calculatethe 3D SDFs using the A model are given in the present paper.The theoretical analyses andexperimental tests have demonstrated that the A model reduces the systematical error radical-ly.It is nearly as simple as the so far used S model,but gives much better results in repro-ducing of 3D SDFs from the measured ID or 2D SDFs.

STEREOLOGICAL CHARACTERIZATION OF MICROSTRUCTURAL EVOLUTION AT LATE STAGES OF SINTERING FOR NICKEL POWDER COMPACTS

The evolution of both the metric and topological properties of the microstructure for Ni pow-der compacts at the late stages of sintering(V_v^s】0.90)has been investigated by means ofstereological method.The effects of precompaction pressure and loose sintering have been stu-died and discussed.

Effects of intraocular rifampicin on retinal ganglion cell structure:a stereological and histopathological study

AIM:To determine the histopathological changes of rifampicin applied intravitreally on retinal ganglion cells by means of stereological and histopathological methods.METHODS:For this study twenty-four New Zealand adult rabbits were divided into four groups(n=6 for each group).50μg/0.1mL(group 1),100μg/0.1mL(group 2),150μg/0.1mL(group 3) and 200μg/0.1mL(group 4),rifampicin were injected into the vitreous of the right eyes of animals,their left eyes were used as control(group 5).After the 28thday of application,animals were anesthetised with xylazine(8mg/kg,IM) and then their eyes were enucleated immediately.Patterns were taken away and eyes were prepared for both stereological and electromicroscopic observation.RESULTS:Depending on the high dose of rifampicin,some histopathological changes such as cytoplasmic dilatation and damaged membrane were observed on the electromicroscopic level.Using quantitative examination,which was done at the light microscopic level,it was shown that the number of neurons decreased linearly as rifampicin dose increased when compared with the control group.CONCLUSION:Based on these findings,low-dose rifampicin(50μg/0.1mL) may be useful for treatment of the ocular diseases.

Stereological study of quantitative relationships between morphological and functional changes of the lungs in early phase of blast injury in rats

The morphological and functional changes of the lungs in the early phase of blast injurywere analyzed and related quantitatively in rats with microcomputer-assisted morphometry.It wasfound that（1）significant or partial correlation exists between stereological and functional indices,（2）changes of the endothelium and epithelium rather than the interstitium contribute to the increase ofharmonic and arithmetic thickness(Tht,Tat)of the air-blood barrier,and（3）the regression equa-tions are as follows:（a）PaO2=3.56+1.62DL（O2）,r=0.678,P=0.032:（b）PaO2=2.99+3.49DL（O2）-0.0005SA+0.003Sc-38.2Vc-0.257Tap-0.028Dt（O2）,ry=0.9999,P=0.008;（c）PaCO2=7.69+0.0001SA-4.35Tat+19.7Tht-46.2Thp-4.4Taen-5.87Taep,ry=0.9999,P=0.014;（d）pH=3.068+0.0005SA-1.79Tat+12.2Tht-50.8Thp-2.52Taint+0.69DL（O2）,ry=0.9999,P=0.10.The significance and application of stereology in theestablishment of the quantitative relationship between morphological and functional indices are dis-cussed.Mathematical models to relate morphometric parameters to functional changes are possible.

Stereological and cytochemical study on the pathogenesis of hepatitis induced by halothane in rats

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Quantitative Evaluation of Inflammatory Response in Rats Submitted to the Abdominal Wall Surgery Using Peritoneum of Paca Preserved in 300% Supersaturated Solution of Sucrose or 98% Glycerin

This study aimed to quantitative evaluate the implantation of paca’s peritoneum, a new biomaterial option, preserved in 300% supersaturated sugar solution or 98% glycerin on the abdominal wall of Wistar rats. 60 males, weighing between 150 and 200 g, are organized into the following experimental groups: control group (GI);group peritoneum preserved in 300% supersaturated sugar solution (GII);and group peritoneum preserved in 98% glycerin (GIII). Each group had 20 animals. Five rats from each group underwent euthanasia at four different moments: 7th, 15th, 30th, and 60th post-operatory day for histological and stereological evaluations of the graft/native tissue interface. On histological examination, one found out an extensive presence of inflammatory infiltrate at the early periods (7th and 15th day) and an extensive presence of connective tissue at the late moments (30th and 60th day). Through stereological analysis, one found out that the number of mononuclear cells decreased throughout the evaluation moments. One concluded that the paca’s peritoneum as a biological membrane preserved in 98% glycerin, when implanted on the abdominal wall of rats, microscopically allowed one to obtain, through stereological analyses, a lower inflammatory response than the paca’s peritoneum preserved in 300% supersaturated sugar solution.

Evaluation of Characteristics of Non-Metallic Inclusions in P/M Ni-Base Superalloy by Automatic Image Analysis

Non-metallic inclusions,especially the large ones,within P/M Ni-base superalloy have a major influence on fatigue characteristics,but are not directly measurable by routine inspection.In this paper,a method,automatic image analysis,is proposed for estimation of the content,size and amount of non-metallic inclusions in superalloy.The methodology for the practical application of this method is described and the factors affecting the precision of the estimation are discussed.In the experiment,the characteristics of the non-metallic inclusions in Ni-base P/M superalloy are analyzed.

Revisiting Sphere Unfolding Relationships for the Stereological Analysis of Segmented Digital Microstructure Images

Sphere unfolding relationships are revisited with a specific focus on the analysis of segmented digital images of microstructures. Since the features of such images are most easily quantified by counting pixels, the required equations are re-derived in terms of the histogram of areas (instead of diameters or radii) as inputs and it is shown that a substitution can be made that simplifies the calculation. A practical method is presented for utilizing negative number fraction bins (which sometimes arise from erroneous assumptions and/or insufficient numbers of observations) for the creation of error bars. The complete algorithm can be implemented in a spreadsheet. The derived unfolding equations are explored using both linear and logarithmic binning schemes, and the pros and cons of both binning schemes are illustrated using simulated data. The effects of the binning schemes on the stereological results are demonstrated and discussed with reference to their consequences for practical materials characterization situations, allowing for the suggestion of guidelines for proper application of this, and other, distribution-free stereological methods.

Effects of different fragments of tenascin-R on neuron morphology in vitro

To investigate the effects of different tenascin-R fragments on morphological changes of neurons in vitro. Methods: Cell suspension was prepared from E14-15 mouse embryo spinal cords by mechanical dissection and trypsin digestion. The cells were cultured in dishes coated with different bacterial expressed tenascin--R fragments. After being cultured in serum-free medium for 26 h, the cells were fixed and stained by ABC immunocytochemistry method for NSE. The cell number and neurites length were measured by a stereological method using an image analysis system, and the data was analyzed statistically. Results:The cells grew well in the serum-free medium for 26 h. Three types of cells were identified: ①phase-bright cells with single or double neurites; ②phasedark cells with well branching neurites; ③flat cells with 2-4 round vesicles in the cell body and radiotlike neurites. The cell number and the neurites length were influenced by different tenascin-R fragments. It was found that FN1-2 fragment inhibited neurite outgrowth. Conclusion: Different tenascin-R fragments that were used as substrate exert varying effects on cultured neural cells, adhesion or anti-adhension of cells, promotion or inhibition of the growth of neurites. These influences were mediated through receptors on the cell membrane. This study may provide some clues to the search for the search for the different receptors which may play essential roles in neuronal development and plasticity.

Evaluation of Collection and Distribution of Samples for Histological,Stereological Analysis and Cell Culture of Canine Mast Cell Tumors

Objective: To collect high quality, representative tissue material from tumors and manage its distribution to different laboratories. Design: Prospective controlled study. Animals: Thirty-six dogs with mast cell tumors. Procedures: The samples were submitted for the following analyses: stereology;histopathology;cell culture;breakdown for cytogenetic analysis of chromosomes (based on the Boxer breed published genome);Cell lysis for Real Time PCR and quantification of gene expression of CX 43, 32 and E-cadherin in canine mast cells. Results: Cytogenetic chromosome analysis, 90.9% of the samples were considered to be of good quality. For gene expression quantification of CX 43, 32 and E-cadherin in canine mast cell tumors (MCT), 95.5% of samples were considered to be of good quality. Conclusions and Clinical Relevance: We seek to assess the importance of surgical collection and post-surgical tissue preparation on laboratory testing by collecting surgical material appropriately to allow accurate diagnosis and reliable clinical prognosis and minimize errors caused by inadequate preparation of samples.

Development and Calibration of a Quantitative,Automated Mineralogical Assessment Method Based on SEM-EDS and Image Analysis:Application for Fine Tailings

Quantitative mineralogy has seen significant developments from the combination of scanning electron microscopy (SEM) with automatic image analysis and energy dispersive X-ray spectrometry (EDS). The mining industry is one of the fields that has benefited from this progress. In this paper, the authors present a newly developed quantitative method based on SEM-EDS and image analysis (IA), which is used to determine the mineralogical and environmental characteristics of mine tailings. The main objectives of the method are to be able to characterize sulphides and carbonates as monomineral particles, which control the acid generation from the tailings. Pure sulphides, calcite and quartz were blended to make mineralogical standards that represent typical mine tailings environmental behavior. The SEM-EDS-IA method achieved good mineralogical precision for medium (1-20 Wt%) and abundant (> 20 Wt%) minerals, with a relative error below 10 %. However, some corrections had to be applied to account for typical stereological effects (apparent particle diameter from polished surface) and preparation modes (particle segregation during resin hardening). Particle size analysis was used to calibrate the method and identify the corrections to be applied. Since mineralogical quantifications are based on the area of the observed particles, the most reliable particle size analyses (also obtained from particle area) typically lead to the best mineralogical characterization. However, the SEM based techniques may show some limitations for fine-grained particle quantification (< 10 μm), which required additional corrections. In this article, the technique is described, and it is applied to characterize fine-grained mine tailings with a size-by-size mineralogy (with sulphides and carbonates content). These results have been used by the Authors to propose an environmental management strategy for acid generating tailings using desulphurization by flotation.

Histo-morphometric vicissitudes in diet-controlled diabetic placenta:A quantitative stereological model

Background:An affordable approach accepted worldwide to successfully treat mild maternal hyperglycemia in gestational diabetes(GDM),is diet-controlled therapy.As no elaborate research was available,this study was crafted to investigate morphometric stereological details and to determine the mean placental oxygen diffusion capacity for patients who were kept on diet therapy for mild gestational hyperglycemia.Methods:A clinical trial(NCT04907708)was conducted from January 2018 to February 2019.A total of 70 women completed the study,out of which 35 served as healthy controls as Group A and 35 were diagnosed as mild gestational diabetics according to WHO criteria(Group B).These patients were kept on a restricted calorie diet with light exercise during gestation and were followed regularly during gestation.Soon after delivery,conserved placentas underwent complete gross,microscopic,and stereological investigations with the point and intersection-counting methods.Results:Significant differences were observed for placental width and syncytial knots(p<0.010 and 0.025 respectively)between the groups on gross and light microscopy.Most of the parameters were non-significant,though numerically more in the GDM group.On stereological details,mean placental volume,mean placental components volumes(villi,inter-villous space,fetal capillary,and fetal connective tissue),mean villi and mean fetal capillary diameter,mean villi and capillary surface density and mean morphometric diffusing capacity of placenta showed non-significant results between the groups.Conclusion:Minimal changes were observed in gross,microscopy,and morphometric stereological details in the placentae of GDM patients managed with nutritional therapy during gestation compared with the healthy controls.