Application of laser-capture microdissection to analysis of gene expression in the testis

The isolation and molecular analysis of highly purified cell populations from complex, heterogeneous tissues has been a challenge for many years. Spermatogenesis in the testis is a particularly difficult process to study given the unique multiple cellular associations within the seminiferous epithelium, making the isolation of specific cell types difficult. Laser-capture microdissection (LCM) is a recently developed technique that enables the isolation of individual cell populations from complex tissues. This technology has enhanced our ability to directly examine gene expression in enriched testicular cell populations by routine methods of gene expression analysis, such as real-time RT-PCR, differential display, and gene microarrays. The application of LCM has however introduced methodological hurdles that have not been encountered with more conventional molecular analyses of whole tissue. In particular, tissue handling (i.e. fixation, storage, and staining), consumables (e.g. slide choice), staining reagents (conventional H&E vs. fluorescence), extraction methods, and downstream applications have all required re-optimisation to facilitate differential gene expression analysis using the small amounts of material obtained using LCM. This review will discuss three critical issues that are essential for successful procurement of cells from testicular tissue sections; tissue morphology, capture success, and maintenance of molecular integrity. The importance of these issues will be discussed with specific reference to the two most commonly used LCM systems; the Arcturus PixCell IIe and PALM systems. The rat testis will be used as a model, and emphasis will be placed on issues of tissue handling, processing, and staining methods, including the application of fluorescence techniques to assist in the identification of cells of interest for the purposes of mRNA expression analysis.     

Novel approaches to analyse glomerular proteins from smallest scale murine and human samples using DIGE saturation labelling

Loss of renal function is often associated with the injury of kidney glomeruli. It is therefore necessary to understand the mechanisms leading to progressive glomerular diseases; this may be addressed using proteomics. Until now, however, analysis of the glomeruli proteome using 2-DE has been technically hampered by low protein yields from scarce samples. To circumvent this problem, we developed a procedure which allows the human and mouse glomeruli proteome to be analysed. In this study, two different approaches were used to isolate mouse and human glomerular protein from kidney cortex. Mouse glomeruli were extracted by embolisation magnetic beads into the glomerular capillaries. Laser capture microdissection (LCM) was utilised to harvest glomeruli from human biopsy material. Human and murine samples were analysed using a fluorescence saturation labelling technique. Using 3 microg mouse glomerular protein a total of 2900 spots were resolved for differential proteome analysis. Moreover, it was also demonstrated for the first time that only ten glomeruli (0.5 microg) picked by LCM from a slide of a human kidney biopsy material were sufficient to visualise 900 spots. This novel strategy paves the way for future experiments aimed at investigating functional proteomics of glomerular diseases in humans and in mice.     

Tissue microdissection techniques in quantitative genome and gene expression analyses

Current advances in quantitative genome and gene expression analyses allow precise molecular genetic fingerprinting of tumor tissues. A crucial factor for the reliability of the data obtained with these refined techniques is the use of morphologically well-defined cell populations. Microdissection technology has been developed to procure pure cell populations from specific areas of tissue sections under microscopic control. This review covers techniques of tissue microdissection in the context of commonly used methods of quantitative genome and gene expression analysis. The first part of the review will summarize the technical aspects of various methods developed for tissue microdissection. In the latter part, current applications of quantitative genome and gene expression analysis techniques employed in microdissected tissue samples will be described.     

Demonstration of 2 distinct glomerular populations by a sieving technic in the kidney cortex of the pig

Amelioration of sieving technics for glomeruli isolation aims at obtention of purer and more homogeneous preparations, presenting a high preservation degree for glomerular functional studies. We have recently demonstrated the necessity to use sieves adapted to the glomeruli size of different considered animals as well as to use kidneys having exactly the same weight in order to obtain very homogeneous glomeruli suspensions. This study presents a new amelioration in the homogeneity of the glomeruli diameter through a preliminary dissection of the renal cortex in order to isolate glomeruli situated in the same particular cortical zone. Pig renal cortex, because of its size, agrees well with a fine dissection in order to dissociate the superficial and the juxta-medullary zones. Glomeruli are isolated with 160/120 micro mesh sieves. Diameter mean value is 218.8 +/- 30.1 micro in superficial glomeruli and 270.4 +/- 30.1 micro in juxta-medullary ones, highly significant difference, (p less than 0.001). Moreover, repartition frequency histograms of the juxta-medullary glomerular populations diameter presents a large shifting to higher values. This renal dissection preceding the glomeruli isolation sieving technique contributes to better the homogeneity of the isolated glomeruli suspensions and opens the way to the original study of the comparative in vitro vasoreactivity of these two different glomerular populations after incubation with physiological or pharmacological reagents.     

Expression microdissection adapted to commercial laser dissection instruments

Laser-based microdissection facilitates the isolation of specific cell populations from clinical or animal model tissue specimens for molecular analysis. Expression microdissection (xMD) is a second-generation technology that offers considerable advantages in dissection capabilities; however, until recently the method has not been accessible to investigators. This protocol describes the adaptation of xMD to commonly used laser microdissection instruments and to a commercially available handheld laser device in order to make the technique widely available to the biomedical research community. The method improves dissection speed for many applications by using a targeting probe for cell procurement in place of an operator-based, cell-by-cell selection process. Moreover, xMD can provide improved dissection precision because of the unique characteristics of film activation. The time to complete the protocol is highly dependent on the target cell population and the number of cells needed for subsequent molecular analysis.     

Expression and Localization of Angiogenic Growth Factors in Developing Porcine Mesonephric Glomeruli

The development and growth of renal glomeruli is regulated by specific angiogenic growth factors, including vascular endothelial growth factor (VEGF) and the angiopoietins (ANGPT1 and ANGPT2). The expression of these factors has already been studied during metanephric glomerulogenesis, but it remains to be elucidated during the development of the embryonic mesonephros, which can function as an interesting model for glomerular development and senescence. In this study, the presence of the angiogenic growth factors was studied in developing porcine mesonephroi, using IHC and real-time RT-qPCR on laser capture microdissected glomeruli. In addition, mesonephric glomerular growth was measured by using stereological methods. ANGPT2 remained upregulated during maturation of glomeruli, which may be explained by the continuous growth of the glomeruli, as observed by stereological examination. The mRNA for VEGFA was expressed in early developing and in maturing glomeruli. The VEGF receptor VEGFR1 was stably expressed during the whole lifespan of mesonephric glomeruli, whereas VEGFR2 mRNA was only upregulated in early glomerulogenesis, suggesting that VEGFR2 is important for the vascular growth but that VEGFR1 is important for the maintenance of endothelial fenestrations. (J Histochem Cytochem 58:1045–1056, 2010)     

Needle in a haystack: microdissecting the proteome of a tissue

Summary. Laser-assisted microdissection is a recent technology that enables cells to be harvested from tissue sections. Proteins can be extracted from the dissected cells for molecular analysis. This enables the analysis of proteins in specific cell types in an in vivo system. Although quantities of protein obtained from the dissected material can be small, it is possible to use established methods such as Western Blotting and 2D-PAGE, as well as newer technologies such as SELDI-MS, to analyse the proteins. This review describes the applications and technical considerations for using laser-assisted dissected cells in proteomics research.     

Laser-assisted microdissection of the kidney: Fundamentals and applications

Laser-assisted microdissection (LAM) permits the procurement of relatively pure cell populations from histological sections. When applied to the kidney, LAM combined with molecular biological techniques has expanded our understanding of renal biology and pathology. Both frozen and fixed renal tissues can be microdissected. However, sample type and tissue processing can influence the quality of molecular data generated. Data analysis may also be complicated by relative variations in gene expression levels. Importantly, preliminary studies have shown that molecular data obtained following LAM on the kidney can offer new diagnostic and prognostic information. Thus, LAM and molecular markers may eventually become incorporated into the routine kidney biopsy examination.     

The long pentraxin PTX3 is synthesized in IgA glomerulonephritis and activates mesangial cells

The long pentraxin PTX3 has been recently involved in amplification of the inflammatory reactions and regulation of innate immunity. In the present study we evaluated the expression and role of PTX3 in glomerular inflammation. PTX3 expression was investigated in the IgA, type I membranoproliferative, and diffuse proliferative lupus glomerulonephritis, which are characterized by inflammatory and proliferative lesions mainly driven by resident mesangial cells, and in the membranous glomerulonephritis and the focal segmental glomerular sclerosis, where signs of glomerular inflammation are usually absent. We found an intense staining for PTX3 in the expanded mesangial areas of renal biopsies obtained from patients with IgA glomerulonephritis. The pattern of staining was on glomerular mesangial and endothelial cells. Scattered PTX3-positive cells were also detected in glomeruli of type I membranoproliferative glomerulonephritis. The concomitant expression of CD14 suggests an inflammatory origin of these cells. Normal renal tissue and biopsies from patients with the other glomerular nephropathies studied were mainly negative for PTX3 expression in glomeruli. However, PTX3-positive cells were detected in the interstitium of nephropathies showing inflammatory interstitial injury. In vitro, cultured human mesangial cells synthesized PTX3 when stimulated with TNF-alpha and IgA and exhibited specific binding for recombinant PTX3. Moreover, stimulation with exogenous PTX3 promoted mesangial cell contraction and synthesis of the proinflammatory lipid mediator platelet-activating factor. In conclusion, we provide the first evidence that mesangial cells may both produce and be a target for PTX3. The detection of this long pentraxin in the renal tissue of patients with glomerulonephritis suggests its potential role in the modulation of glomerular and tubular injury.     

哺乳动物单细胞研究技术的现状与未来

近年来,生命科学和医学的基础研究已深入到单细胞阶段。单细胞研究为揭示生命活动的基本规律、探索细胞异质性、提高对疾病发病机制的认识等提供了重要的线索和依据,同时,单细胞技术已被应用于日常实践中,如法医学和临床生殖医学。单细胞研究中使用的技术也在不断变化,并越来越复杂。文中主要介绍单细胞分离技术,包括手工挑取、激光捕获显微切割和微流控技术,以及单细胞中DNA、RNA和蛋白质分析方法的各种技术。此外,文中总结了近年来生命科学和医学领域的主要单细胞研究成果,讨论了单细胞相关技术和研究的不足,并介绍了其未来的发展方向。     

激光微束在遗传操作上的应用

本文概述了近年来激光微束在染色体微切割和微分离、分子细胞生物学、去除细胞壁、诱导原生质体融合等方面的应用,并对其今后的应用前景作了展望。     

Effect of Immunohistochemistry on Molecular Analysis of Tissue Samples: Implications for Microdissection Technologies

Laser-based tissue microdissection is an important tool for the molecular evaluation of histological sections. The technology has continued to advance since its initial commercialization in the 1990s, with improvements in many aspects of the process. More recent developments are tailored toward an automated, operator-independent mode that relies on antibodies as targeting probes, such as immuno–laser capture microdissection or expression microdissection (xMD). Central to the utility of expression-based dissection techniques is the effect of the staining process on the biomolecules in histological sections. To investigate this issue, the authors analyzed DNA, RNA, and protein in immunostained, microdissected samples. DNA was the most robust molecule, exhibiting no significant change in quality after immunostaining but a variable 50% to 75% decrease in the total yield. In contrast, RNA in frozen and ethanol-fixed, paraffin-embedded samples was susceptible to hydrolysis and digestion by endogenous RNases during the initial steps of staining. Proteins from immunostained tissues were successfully analyzed by one-dimensional electrophoresis and mass spectrometry but were less amenable to solution phase assays. Overall, the results suggest investigators can use immunoguided microdissection methods for important analytic techniques; however, continued improvements in staining protocols and molecular extraction methods are key to further advancing the capability of these methods.     

Testosterone-dependent changes in vivo and in vitro in the structure of the renal glomeruli of the teleost Gasterosteus aculeatus L.

Summary During the reproductive period of the male stickleback, structural and functional changes of the kidney take place, both in the glomeruli and in the renal tubule cells. The structural changes in the glomeruli involve almost all glomerular components and point to a reduction of the glomerular filtration rate. The purpose of this study was to investigate whether these changes are controlled by testosterone, indirectly or directly. In vivo experiments demonstrated that exposure of immature castrates for 8 days to methyltestosterone leads to activation of mesangial cells and podocytes, to slight expansion of mesangial matrix and slight thickening of basal lamina. Observations on cultured renal tissue showed that two androgens, 11-ketotestosterone and methyl testosterone, are also able to stimulate the secretory activity of podocytes and mesangial cells in vitro. The results therefore indicate, that most glomerular changes in male sticklebacks during the reproductive season are directly effected by testosterone.