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.

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.

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.

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.

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.

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

This study was designed to investigate the relationship between hepatotoxicity of halothane and altered hepatic calcium homeostasis in enzyme-induced hypoxic rats by means of Ca2+ cytochemical location and EDX microanalysis combined with stereology of liver and ultrastructural analysis by computer. It was found that more calcium precipitated in the cytoplasm and mitochondria of liver than that in the controls following halothane exposure. This alteration appears to be the result of accelerated uptake of Ca2+ by hepatocytes and decreased output of loaded Ca2+ on the one hand, and increased release of reserved Ca2+ on the other hand. These results suggest that change in cullular Ca2+ balance plays an important role in the mechanism of hepatotoxicity induced by halothane exposure.

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.

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.

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.

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.

米诺环素对脊神经结扎大鼠脊髓内M1/M2型小胶质细胞极化的影响

目的探讨米诺环素(minocycline,Mino)对脊神经结扎(spinal nerve ligation,SNL)致神经病理性疼痛(neuropathic pain,NP)大鼠脊髓内M1型(促炎型)和M2型(抗炎型)小胶质细胞极化的影响及其机制。方法36只成年雄性SD大鼠按体质量分层随机抽样分为Sham组、SNL组和M+SNL组(鞘内注射米诺环素),于术前1 d和术后14 d内测定大鼠后足机械痛阈值和冷痛敏阈值。术后14 d于L5脊髓取材,采用免疫组化技术和体视学技术检测L5脊髓背角内小胶质细胞的总数以及M1/M2型小胶质细胞的数量;利用生物信息学技术筛选出脊髓小胶质细胞内的核心靶点Cst7,并采用Western blot检测小胶质细胞标志物Iba-1、M1型小胶质细胞标志物iNOS、M2型小胶质细胞标志物CD206、Cst7基因编码的蛋白质胱抑素F(cystatin F,CF)及CatS/CX3CL1/CX3CR1通路蛋白的表达水平;采用ELISA检测脊髓内TNF-α、IL-6和IL-10的蛋白表达量。结果M+SNL组大鼠术后7~14 d手术侧的机械痛和冷痛敏阈值均比SNL组显著升高(P<0.01)。SNL组大鼠手术侧脊髓背角内小胶质细胞的总数,M1型、M2型小胶质细胞的数量以及脊髓CatS、CX3CL1、CX3CR1、TNF-α、IL-6和IL-10蛋白表达较Sham组均显著增加,而CF含量显著降低(P<0.01);与SNL组比较,M+SNL组小胶质细胞的总数、M1型小胶质细胞的数量以及CatS、CX3CL1、CX3CR1、TNF-α、IL-6蛋白表达均显著减少,而M2型小胶质细胞的数量以及CF、IL-10的蛋白表达均显著增加(P<0.05)。结论米诺环素改善SNL致神经病理性疼痛的机制可能是通过上调小胶质细胞内CF蛋白表达,进而抑制CatS/CX3CL1/CX3CR1信号通路,促进小胶质细胞由M1型向M2型转化,以平衡M1/M2小胶质细胞极化失衡、减轻神经炎症。

矿物表面解离度分析技术发展综述

矿物解离程度显著影响浮选分离效率,矿物在颗粒表面解离行为的精准解析是实现浮选过程高效调控的前提。总结了矿物加工领域常见的矿物表面解离度分析方法:基于扫描电子显微镜(SEM)的2D解离度分析、2D解离度的体视学修正和3D解离度分析方法(X射线显微断层扫描成像技术)。2D解离度分析方法应用于低解离度粗粒连生体时存在体视学偏差,并且复杂共伴生颗粒的2D矿物解离度体视学修正仍面临挑战。2D解离分析适用于矿物单体解离或者高度解离的传统浮选处理的细颗粒,而对于矿物解离度较低的粗颗粒,3D解离度分析不存在体视学偏差,可提供更准确的表面解离度分析结果。

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.

跑步对抑郁大鼠内侧前额叶皮质兴奋性突触的影响

目的:精确定量研究跑步锻炼对慢性束缚应激(chronic restraint stress,CRS)诱导的抑郁模型大鼠内侧前额叶皮质(medial prefrontal cortex,mPFC)内Sp^(+)兴奋性突触数量的影响。方法:选取雄性SD大鼠(54只),经过适应性喂养和糖水基线调整,在CRS模型建立成功后随机分为对照组、抑郁模型组和模型跑步组,其中模型跑步组大鼠在束缚的第5周开始进行为期4周的跑步锻炼干预。最后,对各组大鼠进行行为学测试,并运用免疫组织化学技术结合现代体视学方法对各组大鼠mPFC内Sp^(+)兴奋性突触变化进行精确定量研究。结果:与对照组[(97.14±2.64)%]相比,抑郁模型组和模型跑步组大鼠糖精偏好百分比[(89.62±6.05)%]减少(P=0.002),体质量的增长减缓,强迫游泳实验中大鼠的不动时间和新环境进食抑制实验的进食潜伏期增加。4周的跑步锻炼可以有效减缓抑郁模型组大鼠糖精偏好百分比的下降[(89.30±5.06)%vs.(97.30±2.08)%,P=0.018],降低强迫游泳实验中抑郁大鼠的不动时间,并在新环境进食抑制实验中缩短抑郁大鼠的进食潜伏期。体视学精确定量分析结果显示,抑郁模型组大鼠mPFC内的Sp^(+)兴奋性突触总量[(9.98±0.35)×10^(8)个]低于对照组[(11.50±1.27)×10^(8)个,P=0.013]。而跑步锻炼则可以逆转抑郁大鼠mPFC内的Sp^(+)兴奋性突触总数的减少[模型跑步组(11.30±1.21)×10^(8)个,P=0.003]。结论:跑步锻炼干预后CRS抑郁模型大鼠mPFC的Sp^(+)兴奋性突触数量的改变可能是跑步锻炼发挥抗抑郁作用的神经生物学基础之一。

Stereological Analysis of Peritubular Cells in Aging Mouse Testes

Objective To analyse quantitative changes of testicular peritubular cells in aging mice, and meanwhile investigate age-related changes in both Sertoli cells and germ cells with stereology. Methods Thirty male Kunming mice were enrolled in this study. The aged mice （n=15） were 18 months old. The young mice （n=15） were 6 months old, as the control. Haematoxylin and eosin staining was used to observe histological changes. Pathological evaluation rules of assignment scores were made to evaluate. Stereology techniques were applied to quantitatively analyse histological structure of the testicular tissues. Results Compared with young group, pathologic assignment scores of aging group were significantly higher （P〈0.05）. Diameter of seminiferous tubules in aging mice was atrophied significantly （P〈0.05）. Germ cell numerical density and Sertoli cell volume density of aging group were reduced significantly compared with young group （P〈0.05）. No significant differences were found in peritubular cell parameters compared with young group （P〉0.05）. Conclusion The pathological assignment scores suggest a succession of changes on the testis with age. The stereological numbers demonstrate damaged Sertoli cells and decreased germ cells, which is indicative of spermatogenic disturbance. However, no significant quantity changes of peritubular cells is observed in aging mice.