Application of Stereology on Neodymium-Doped Yttrium Aluminum Garnet (Nd∶YAG) Transparent Ceramics

Nd ： YAG precursor powders were synthesized by homogeneous precipitation, and Nd ： YAG transparent ceramics were prepared by vacuum sintering at 1700 ℃ for 5 h. The ceramic materials were characterized by light transmittance and field emission gun-environment scanning microscope. Using statistics and stereology theory, study was carried out on the quantitative relationships between light transmittance and stereological parameters in three-dimensional Euclidean space. It is found that the transmittance of Nd：YAG with 1 mm in thickness is about 45% and 58% in visible and near-infrared wavelength, respectively. The transmittance linearly increases with increasing equivalent sphere diameter and reaches the theoretical value of single crystal when the equivalent sphere diameter is 20μm. The transmittance decreases with the increasing of mean specific area per unit volume of grain and discrete grains, and the transmittance decreases with increasing mean free distance of grains in Nd：YAG ceramics.

Stereology in Materials Science

Quantitative analysis of populations having a geometric structure,which has developed into a special scientific subject called microstructology or stereology,is of great importance to the characterization and evaluation of microstructures and their evolution in various processes.This paper, besides a brief discussion on those topics such as the recent developments of computer assisted image analysis,mathematical morphology,and fractal analysis,will mainly focus on the scope,fundamen- tals,present status,and perspectives of classical stereology.Several case examples of its application to 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.

Histological changes of the testis and epididymis in adult rats as a result of Leydig cell destruction after ethane dimethane sulfonate treatment： a morphometric study

Aim： To quantitatively study the histological changes of the testis and epididymis as a result of a drastic reduction of testosterone secretion. Methods： Fourteen adult Sprague-Dawley rats were injected intraperitoneally with ethane dimethane sulfonate （EDS, 75 mg/kg） and the same number of animals were injected with normal saline as a control. At days 7 and 12 （after treatment）, respectively, half of the animals from each group were killed. The testes and epididymides were removed and tissue blocks embedded in methacrylate resin. The cell number per testis was estimated using the stereological optical disector and some other parameters were obtained using other morphometric methods. Results： The EDS treatment resulted in an almost complete elimination of Leydig cells but had no effect on the numbers of Sertoli cells per testis. At day 7 after EDS treatment, many elongated spermatids were retained in the seminiferous epithelium and many round spermatids could be seen in the epididymal ducts. At day 12, a looser arrangement of spermatids and spermatocytes became evident, with apparent narrow empty spaces being formed between germ cells in an approximately radial direction towards the tubule lumen; the numbers （per testis） of non-type B spermatogonia and spermatocytes were similar to controls, whereas that of type B spermatogonia increased by 59%, and that of early round, elongating and late elongated spermatids decreased by 37%, 72% and 52%, respectively. Conclusion： The primary spermatogenic lesions following EDS administration were （i） spermiation failure and （ii） detachment of spermatids and spermatocytes associated with impairment in spermiogenesis and meiosis.

Quantitative (stereological) study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats

Aim: To evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate (TU) treatment. Methods: Adult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector. Results: In response to TU treatment, the numbers of non-type B spermatogonia, type B spermatogonia and late elongated spermatids per testis were reduced to 51 %, 66 % and 14 % of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51 % - 65 % of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0 % of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged. Conclusion: Double inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gona-dotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular testosterone levels.

Quantitative (stereological) study on the spermatozoal storage capacity of epididymis in rats and monkeys

Aim: To investigate the spermatozoal production rate of the testis and the spermatozoal storage capacity of the epi-didymis in monkeys and rats. Methods: The number of the late spermatids (steps 13 - 14 in the monkey or steps15 - 19 in the rat) per testis and the number of spermatozoa per epididymis were estimated in 6 normal adult monkeys(Macaca fascicularis) and 6 normal adult SD rats on 25 um-thick methacrylate-embedded sections using a contempo-rary unbiased and efficient stereological method-the optical disector. The diameter and length of the efferent ductulesand ductus epididymidis and the volume of the epididymal fluid in the tubules were also estimated. Results: The totalnumber of the late spermatids per testis was 2902 ±749 (million, x ±s) in the monkey, or 179 ±31 in the rat; thenumber of spermatozoa per epididymis was 3235 ±1835 in the monkey, or 241±76 in the rat. Conclusion: A largenumber of spermatozoa was densely packed and stored in the ductus epididymidis; the epididymal transit time for sper-matozoa was around 5 days in monkeys or 11 days in rats.

Reversible effect of testosterone undecanoate injection on spermatogenesis in rats

Aim: To study the effect of testosterone undecanoate (TU) injection on spermatogenesis in rats. Methods:Twenty adult SD rats received vehicle or TU (8 mg/kg, 19 mg/kg or 625 mg/kg) injection, im, every 15 days for 60days, and another 38 animals received similar treatments for 130 days with half of them undergoing a recovery phase of120 days (5 rats for each treatment). At the end of the treatment, testes were removed and the diameter of the seminif-erous tubules and the number of late elongated spermatids (steps 15-19) per testis were estimated with stereologicalmethods as a measure of the spermatogenic efficiency. Results: Low dose (8 mg/kg) TU treatment virtually hadno effect on spermatogenesis. A dose of 19 mg/kg slightly suppressed spermatogenesis 60 days after treatment, and se-vere suppression occurred after another 70 days of dosing. Spermatogenesis was completely recovered at the end of therecovery phase. Large dose (625 mg/kg) TU treatment did not significantly affect spermatogenesis and was well toler-ated by animals. Conclusion: TU injection reversibly suppresses spermatogenesis in rats.

Quantitative characterization of β-solidifying γ-TiAl alloy with duplex structure

For precise plastic deformation,microstructure control is essential especially for β-solidifying γ-TiAl alloy with duplex structure.Based on stereology,the microstructure of isothermally compressed γ-TiAl alloy was divided into β_(0) grains,remnant α_(2)/γ lamellar colonies,α_(2) and γ grains.The results show that the volume fraction of β_(0) grains slightly increases in the isothermally compressed γ-TiAl alloy with the increase of height reduction.Meanwhile,the volume fractions of remnant α_(2)/γ lamellar colonies and α_(2) grains decrease.However,the volume fraction of γ grains increases from 64.39% to 78.47%.According to the quantitative results,the α→γ phase transformation was investigated in-depth,and it is found that isothermal compression accelerates the α→γ phase transformation.The first α→γ phase transformation is similar to ledge-controlled transformation,through which remnant α_(2)/γ lamellar colonies finally convert intoγgrains in isothermal compression.The second is achieved by α/γ phase interface immigration.

A quantitative study on changes of the myelinated fibers in the cerebral cortex of cortical dysplasia rats

An animal model of cortical dysplasia was established through X-ray irradiation induced subcortical heterotopic nodules in rats. Transmission electron microscopy detection of the ultrastructure and the stereology examination showed that there was a significant decrease in cerebral white matter and hippocampal volume, the total volume, volume density, length density and total length of the myelinated fibers in the white matter of cortical dysplasia rats. Subcortical heterotopic nodules of the hippocampal CA1 region and synaptic number density in the CA3 region were reduced compared with normal rats. Our experimental findings indicate that erosed subcortical heterotopic nodules, decreased total length of myelinated nerve fibers and demyelination directly lead to a reduction of white matter volume.

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.

Direct muscle neurotization after end-to-end and end-to-side neurorrhaphy An experimental study in the rat forelimb model

The need for the continuous research of new tools for improving motor function recovery after nerve injury is justified by the still often unsatisfactory clinical outcome in these patients. It has been previously shown that the combined use of two reconstructive techniques, namely end-to-side neurorrhaphy and direct muscle neurotization in the rat hindlimb model, can lead to good results in terms of skeletal muscle reinnervation. Here we show that, in the rat forelimb model, the combined use of direct muscle neurotization with either end-to-end or end-to-side neurorrhaphy to reinnervate the denervated flexor digitorum muscles, leads to muscle atrophy prevention over a long postoperative time lapse （10 months）. By contrast, very little motor recovery （in case of end-to-end neurorrhaphy） and almost no motor recovery （in case of end-to-side neurorrhaphy） were observed in the grasping activity controlled by flexor digitorum muscles. It can thus be concluded that, at least in the rat, direct muscle neurotization after both end-to-end and end-to-side neurorrhaphy represents a good strategy for preventing denervation-related muscle atrophy but not for regaining the lost motor function.

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.

Seeing the wood for the trees:towards improved quantification of glial cells in central nervous system tissue

The following mini-review attempts to guide researchers in the quantification of fluorescently-labelled proteins within cultured thick or chromogenically-stained proteins within thin sections of brain tissue.It follows from our examination of the utility of Fiji Image J thresholding and binarization algorithms.Describing how we identified the maximum intensity projection as the best of six tested for two dimensional（2 D）-rendering of three-dimensional（3 D） images derived from a series of z-stacked micrographs,the review summarises our comparison of 16 global and 9 local algorithms for their ability to accurately quantify the expression of astrocytic glial fibrillary acidic protein（GFAP）,microglial ionized calcium binding adapter molecule 1（IBA1） and oligodendrocyte lineage Olig2 within fixed cultured rat hippocampal brain slices.The application of these algorithms to chromogenically-stained GFAP and IBA1 within thin tissue sections,is also described.Fiji’s Bio Voxxel plugin allowed categorisation of algorithms according to their sensitivity,specificity accuracy and relative quality.The Percentile algorithm was deemed best for quantifying levels of GFAP,the Li algorithm was best when quantifying IBA expression,while the Otsu algorithm was optimum for Olig2 staining,albeit with over-quantification of oligodendrocyte number when compared to a stereological approach.Also,GFAP and IBA expression in 3,3′-diaminobenzidine（DAB）/haematoxylin-stained cerebellar tissue was best quantified with Default,Isodata and Moments algorithms.The workflow presented in Figure 1 could help to improve the quality of research outcomes that are based on the quantification of protein with brain tissue.

Stereological method for objectively quantifying myelin sheaths in the rat hippocampus

In the present study,tissue blocks were randomly sampled from the entire hippocampus of 6-week-old Long-Evans rats.Isotropic,uniform and random sections,60 nm thick,were prepared by isector.Fifteen fields of view were randomly selected for each section and photographed using a transmission electron microscope.The mean internal and external diameters of the myelin sheaths were obtained by measuring the longest profile diameter perpendicular to its longest axis.The inner and outer perimeters of the myelin sheaths were estimated using the equidistant parallel test lines.The thickness of the myelin sheaths was estimated by direct orthogonal measurements in uniform,random locations.These stereological methods should permit an unbiased quantitative assessment of changes in the myelin sheaths of myelinated fibers in the hippocam-pus.

Stereological study of the effects of vitamin E on testis structure in rats treated with para-nonylphenol

This study was organized to see whether vitamin E, as a strong antioxidant, could affect the abnormalities of testis structure caused by para-nonylphenol （p-NP） during its development. A total of 32 female Wistar rats after mating were divided into four groups （n = 8）： control, vitamin E （100 mg kg^-1 per day）, p-NP （250 mg kg^-1 per day） and p-NP ＋ vitamin E. The rats were treated from the seventh day of pregnancy till the twenty-first day. After weaning, the male pups were divided into the same groups and were treated orally for 90 days. Finally, the right testis was fixed, processed, stained and studied using stereological methods. The weight and volume of testis, volume of seminiferous tubules and its diameter, thickness of the basement membrane, height of the germinal epithelium, total number of types A and B spermatogonia, spermatocyte, spermatid and Sertoli cells were significantly reduced in p-NP group when compared with other groups. Co-administration of vitamin E and p-NP compensated for the adverse effects of p-NP on the above parameters. In addition, treatment with only vitamin E caused a significant increase in diameter, basement membrane thickness and height of germinal epithelium, number of spermatogonia and spermatocytes. Co-administration of vitamin E with p-NP could prevent the adverse effects ofp-NP on the testis structure during its development.

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 of stereological method.The effects of precompaction pressure and loose sintering have been stu- died and discussed.

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.The origin is the basic supposition of spherical grains in the S model,which does not correspond with 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 calculate the 3D SDFs using the A model are given in the present paper.The theoretical analyses and experimental 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 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.