LMD and LC-MS-based chemical constituents and pharmacological effects assessment for two different processing methods of the root of Paeonia lactiflora Pall.

The plant of Paeonia lactiflora Pall. belongs to Ranunculaceae, and its root can be divided into two categories according to different processing methods, which included that one was directly dried without peeling the root of the P. lactiflora (PR), and the other was peeled the root of the P. lactiflora (PPR) after boiled and dried. To evaluate the difference of chemical components, UPLC-ESI-Q-Exactive Focus-MS/MS and UPLC-QQQ-MS were applied. The distribution of chemical components in different tissues was located by laser microdissection (LMD), especially the different ingredients. A total of 86 compounds were identified from PR and PPR. Four kind of tissues were isolated from the fresh root of the P. lactiflora (FPR), and 54 compounds were identified. Especially the content of gallic acid, albiflorin, and paeoniflorin with high biological activities were the highest in the cork, but they were lower in PR than that in PPR, which probably related to the process. To illustrate the difference in pharmacological effects of PR and PPR, the tonifying blood and analgesic effects on mice were investigated, and it was found that the tonifying blood and analgesic effects of PPR was superior to that of PR, even though PR had more constituents. The material basis for tonifying blood and analgesic effect of the root of P. lactiflora is likely to be associated with an increase in constituents such as paeoniflorin and paeoniflorin lactone after boiled and peeled. The study was likely to provide some theoretical support for the standard and clinical application.

A comprehensive assessment of predictors of fertility outcomes in men with non-obstructive azoospermia undergoing microdissection testicular sperm extraction.

BACKGROUND: Microdissection testicular sperm extraction (microTESE) in men with non-obstructive azoospermia (NOA) is the procedure that results in the highest number of sperm cells retrieved for in vitro fertilization (IVF). This study presents a novel assessment of predictors of sperm retrieval as well as downstream embryology and pregnancy outcomes in cases of men with NOA undergoing microTESE. METHODS: A retrospective chart review of 72 men who underwent microTESE for predictors of fertility outcomes including sperm retrieved at microTESE, embryology progression to embryo transfer (ET), clinical pregnancy, live birth, and surplus sperm retrieved for additional IVF/intracytoplasmic injection cycles beyond one initial cycle. Statistical models for each of these outcomes were fitted, with a p-value of < 0.05 considered significant for the parameters estimated in each model. RESULTS: Seventy-two men underwent microTESE, and 51/72 (70.8%) had sperm retrieved. Of those, 29/43 (67.4%) reached ET. Of the couples who underwent ET, 21/29 (72.4%) achieved pregnancy and 18/29 (62.1%) resulted in live birth. Of the men with sperm retrieved, 38/51 (74.5%) had surplus sperm cryopreserved beyond the initial IVF cycle. Age, testicular volume, FSH, and testicular histopathology were assessed as predictors for sperm retrieved at microTESE, progression to ET, pregnancy, live birth, and surplus sperm. There were no preoperative predictors of sperm retrieval, clinical pregnancy, or live birth. Age predicted reaching ET, with older men having increased odds. FSH level had a negative relationship with surplus sperm retrieved. Men with hypospermatogenesis histology had higher rates of sperm retrieval, clinical pregnancy, live birth, and having surplus sperm. CONCLUSIONS: Men who underwent microTESE with a hypospermatogenesis histopathology had better outcomes, including higher rates of sperm retrieval, clinical pregnancy, live birth, and having surplus sperm retrieved. Increasing male partner age increased the odds of reaching ET. No other clinical factors were predictive for the outcomes considered.

Intraoperative assessment of tubules in predicting microdissection testicular sperm extraction outcome in men with Sertoli cell-only syndrome.

OBJECTIVE: This study aimed to assess the value of measuring the tubule diameter during microdissection testicular sperm extraction (micro-TESE) in predicting outcomes in patients with Sertoli cell-only syndrome (SCOS). METHODS: Fifty-six consecutive patients with SCOS were included. Patients were classified into two groups on the basis of the diameter of seminiferous tubules measured against 5/0 surgical suture (≥100 µm or <100 µm). RESULTS: The sperm retrieval rate (SRR) in men with a tubule diameter ≥100 µm was significantly lower than that in those with <100 µm (3.1% vs. 25.0%). The SRR from the contralateral testis in men with a tubule diameter ≥100 µm was lower than that in those with <100 µm (0% vs. 14.3%). Men with a tubule diameter ≥100 µm had a significantly larger testis and lower follicle-stimulating hormone levels than did men with <100 µm (8.1 ± 2.4 vs. 5.3±1.8 mL, 19.9 ± 9.7 vs. 25.9 ± 7.1 mIU/mL, respectively). CONCLUSIONS: The diameter of tubules is a useful predictor for a successful SRR in men with SCOS. Intraoperative assessment of homogeneous large tubules allows some men to perform a limited (superficial) contralateral micro-TESE after no spermatozoa are initially identified.

Transcriptomic Landscape of von Economo Neurons in Human Anterior Cingulate Cortex Revealed by Microdissected-Cell RNA Sequencing.

The von Economo neurons (VENs) are specialized large bipolar projection neurons with restricted distribution in the human brain, and they are far more abundant in humans than in non-human primates. However, VEN functions remain elusive due to the difficulty of isolating VENs and dissecting their connections in the brain. Here, we combined laser-capture-microdissection with RNA sequencing to describe the transcriptomic profile of VENs from human anterior cingulate cortex (ACC). Using pyramidal neurons as reference cells, we identified 344 genes with VEN-associated expression differences, including 215 higher and 129 lower expression genes. Functional enrichment and protein-protein interaction network analyses showed that these genes with VEN-associated expression differences are involved in VEN morphogenesis and functions, such as dendrite branching and axon myelination, and many of them are associated with human social-emotional disorders. With the use of in situ hybridization and immunohistochemistry assays, we validated four novel VEN markers (VAT1L, CHST8, LYPD1, and SULF2). Collectively, we generated a full-spectrum expression profile of VENs from human ACC, greatly enlarging the pool of genes with VEN-associated expression differences that can help researchers to understand the role of VENs in normal and disordered human brains.

Assessment of the use of contrast enhanced ultrasound in guiding microdissection testicular sperm extraction in nonobstructive azoospermia.

BACKGROUND: The aim of this study is to assess the value of contrast-enhanced ultrasound (CEUS) as a new non-invasive approach to locate the testicular area in which spermatogenesis is most likely to be found in non-obstructive azoospermic testes and to evaluate the accuracy of CEUS as a predictor of successful sperm retrieval. METHODS: CEUS was performed in 120 nonobstructive azoospermia (NOA) patients. Microdissection testicular sperm extraction (M-TESE) was performed on the best and poorest perfusion areas selected by CEUS and on conventional areas. RESULTS: In the 187 testicles that underwent M-TESE, the sperm retrieval rates (SRRs) in the best perfusion area and poorest perfusion area over the maximal longitudinal section and conventional area were 63.1, 34.7 and 47.1%. According to receiver operating characteristic (ROC) analysis, the arrival times (AT) ≤27 s, time-to-peak intensity (TTP) ≤45 s, and peak intensity (PI) ≥11 dB were the best predictors of positive sperm retrieval. The location of the best perfusion area was able to guide M-TESE to improve the success rates. CONCLUSIONS: Testicle CEUS is suggested to be performed in all patients with NOA. If AT≤27 s, TTP ≤ 45 s or PI≥11 dB are found in the best perfusion area, M-TESE is strongly recommended.

Incidental Durotomy in Open Versus Tubular Revision Microdiscectomy: A Retrospective Controlled Study on Incidence, Management, and Outcome.

STUDY DESIGN: Retrospective case-control study. OBJECTIVE: To compare the incidence, management, and outcome of incidental durotomy in revision microdiscectomy with open and minimal-access surgery. SUMMARY OF BACKGROUND DATA: Incidental durotomy occurs with a variable incidence of 3%-27% in spine surgery. The highest rate occurs in revision microdiscectomy. The intraoperative and postoperative management of dural tears varies in the literature and the definite impact on clinical outcome has to be clarified. METHODS: This is a retrospective study of medical records of 135 patients who underwent revision microdiscectomy, divided into 2 subgroups: OPEN (n=82) versus minimal-access surgery (MINI, n=53). Occurrence of intraoperative dural tears, intraoperative and postoperative management of durotomy, and clinical outcomes, according to MacNab criteria, were retrospectively examined. Statistical comparisons for categorical values between groups were accomplished using the 2-tailed Fisher exact test. P-values <0.05 were considered to be statistically significant. RESULTS: The incidence of durotomy in group OPEN was 19.5% (n=16/82) and in group MINI 17.0% (n=9/53) (P=0.822). The majority of durotomies (23/25) were repaired with an absorbable fibrin sealant patch alone. Postoperative cerebrospinal fluid fistula occurred only in 1 case of the OPEN group and was treated with lumbar drainage without the need for a reoperation. Patients with durotomy of the MINI group tended to have better outcome compared with those of the OPEN group without being statistically significant. CONCLUSIONS: The incidence of durotomy and postoperative cerebrospinal fluid fistula in lumbar revision microdiscectomy does not significantly differ between minimal-access and standard open procedures. The application of a fibrin sealant patch alone is an effective strategy for dural repair in revision lumbar microdiscectomy.

Isolation and functional assessment of cutaneous stem cells.

The epidermis and associated appendages of the skin represent a multi-lineage tissue that is maintained by perpetual rounds of renewal. During homeostasis, turnover of epidermal lineages is achieved by input from regionalized keratinocytes stem or progenitor populations with little overlap from neighboring niches. Over the last decade, molecular markers selectively expressed by a number of these stem or progenitor pools have been identified, allowing for the isolation and functional assessment of stem cells and genetic lineage tracing analysis within intact skin. These advancements have led to many fundamental observations about epidermal stem cell function such as the identification of their progeny, their role in maintenance of skin homeostasis, or their contribution to wound healing. In this chapter, we provide a methodology to identify and isolate epidermal stem cells and to assess their functional role in their respective niche. Furthermore, recent evidence has shown that the microenvironment also plays a crucial role in stem cell function. Indeed, epidermal cells are under the influence of surrounding fibroblasts, adipocytes, and sensory neurons that provide extrinsic signals and mechanical cues to the niche and contribute to skin morphogenesis and homeostasis. A better understanding of these microenvironmental cues will help engineer in vitro experimental models with more relevance to in vivo skin biology. New approaches to address and study these environmental cues in vitro will also be addressed.

Novel Cell and Tissue Acquisition System (CTAS): microdissection of live and frozen brain tissues.

We developed a novel, highly accurate, capillary based vacuum-assisted microdissection device CTAS-Cell and Tissue Acquisition System, for efficient isolation of enriched cell populations from live and freshly frozen tissues, which can be successfully used in a variety of molecular studies, including genomics and proteomics. Specific diameter of the disposable capillary unit (DCU) and precisely regulated short vacuum impulse ensure collection of the desired tissue regions and even individual cells. We demonstrated that CTAS is capable of dissecting specific regions of live and frozen mouse and rat brain tissues at the cellular resolution with high accuracy. CTAS based microdissection avoids potentially harmful physical treatment of tissues such as chemical treatment, laser irradiation, excessive heat or mechanical cell damage, thus preserving primary functions and activities of the dissected cells and tissues. High quality DNA, RNA, and protein can be isolated from CTAS-dissected samples, which are suitable for sequencing, microarray, 2D gel-based proteomic analyses, and Western blotting. We also demonstrated that CTAS can be used to isolate cells from native living tissues for subsequent recultivation of primary cultures without affecting cellular viability, making it a simple and cost-effective alternative for laser-assisted microdissection.

A simple, cost-effective and flexible method for processing of snap-frozen tissue to prepare large amounts of intact RNA using laser microdissection.

Understanding the molecular basis of disease requires gene expression profiling of normal and pathological tissue. Although the advent of laser microdissection (LMD) has greatly facilitated the procurement of specific cell populations, often only small amounts of low quality RNA is recovered. This precludes the use of global approaches of gene expression profiling which require sizable amounts of high quality RNA. Here we report a method for processing of snap-frozen tissue to prepare large amounts of intact RNA using LMD. Portions of small intestine from piglets (n = 6) were snap-frozen in Optimum Cutting Temperature compound (experimental) and in RNAlater (control). A randomly selected sample was laser microdissected using the developed protocol in multiple sessions totalling 4 h each day on four consecutive days. RNAs were extracted from these samples and its control and their quality (RIN) determined. RINs of the experimental samples were independent of time (p = 0.12) and day (p = 0.56) of the microdissection thereby suggesting that their RNA quality remained unaltered. These samples exhibited high quality (RIN ≥ 8) with good recovery (81.2%) and excellent yield (1539 ng/1.2 × 10(7) µm(2)). Their overall RIN, 8.029 ± 0.116, was not significantly different from 8.2 (p = 0.123), the value obtained from the control, non-laser microdissected, sample. This indicated that the RNA quality from the laser microdissected and non-microdissected samples was comparable. The method allowed LMD for up to 4 h each day for a total of four days. The microdissected samples can be pooled thereby increasing amount of RNA at least by ten-fold. The procedure did not require any expensive limited-shelf life RNase inhibitors, RNA protectors, staining kits or toxic chemicals. Furthermore, it was flexible and enabled the processing without affecting routine laboratory workflow. The method developed was simple, inexpensive and provided substantial amounts of high quality RNA suitable for gene expression profiling and other cellular and molecular analyses for biology and molecular medicine.

O6-methylguanine-DNA-methyltransferase promoter methylation assessment by microdissection-assisted methylation-specific PCR and high resolution melting analysis in patients with glioblastomas.

We have determined O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status by methylation-specific polymerase chain reaction (MSP) in 22 paraffin-embedded specimens of glioblastoma multiforme. A MGMT methylation-specific high resolution melting (HRM) assay was performed to compare the methylation levels of the tumorous and non-tumorous portions of each sample, which were selectively collected using a microdissection technique. MGMT methylation was detected in 10 patients using MSP, while 8 patients had both methylated and unmethylated MGMT promoters. HRM assays showed that there was no difference in the level of methylation between tumorous and non-tumorous portions of each sample. In patients with MSP-positive tumors, the overall survival (median, 22 months) was longer as compared to those with MSP-negative tumors (median, 14 months). A correlation between the methylation status of the MGMT promoter and MGMT protein expression was observed in 12 samples. This study demonstrates that MGMT methylation is not restricted to glioblastoma cells. Additionally, methylation-specific HRM is a feasible approach that can be readily applied to the methylation analysis of MGMT. A further study will be needed to determine the dynamic change of MGMT methylation in the tumor environment.

Isolation of guard cells from fresh epidermis using a piezo-power micro-dissection system with vibration-attenuated needles.

The Eppendorf Piezo-Power Microdissection (PPMD) system uses a tungsten needle (MicroChisel) oscillating in a forward-backward (vertical) mode to cut cells from surrounding tissue. This technology competes with laser-based dissection systems, which offer high accuracy and precision, but are more expensive and require fixed tissue. In contrast, PPMD systems can dissect freshly prepared tissue, but their accuracy and precision is lower due to unwanted lateral vibrations of the MicroChisel. Especially in tissues where elasticity is high, these vibrations can limit the cutting resolution or hamper the dissection. Here we describe a cost-efficient and simple glass capillary-encapsulation modification of MicroChisels for effective attenuation of lateral vibrations. The use of modified MicroChisels enables accurate and precise tissue dissection from highly elastic material.

mRNA and micro-RNA expression analysis in laser-capture microdissected prostate biopsies: valuable tool for risk assessment and prevention trials.

Diagnosis of prostate cancer (PCa) typically relies on needle biopsies, which are routinely archived in paraffin after formalin fixation and may contain valuable risk or prognostic information. The objective of this study was to determine the feasibility of mRNA and miRNA expression analysis in laser-capture microdissected (LCM) formalin-fixed paraffin-embedded archived prostate biopsies compared to the gold standard of frozen tissue. We analyzed the expression of compartment-specific and PCa-related genes in epithelial and stromal tissues collected from paired sets of archived prostate biopsies and frozen radical prostatectomy specimens from three patients. Our results showed appropriate compartment-specific and PCa-related expression with good within patient agreement between the FFPE biopsies and the frozen tissue. The potential for both mRNA and micro-RNA expression profiling in the biopsies was also demonstrated using PCR arrays which showed high correlation between the biopsy and frozen tissue, notwithstanding sensitivity limitations for mRNA detection in the FFPE specimen. This is the first study to compare RNA expression from biopsy and frozen tissues from the same patient and to examine miRNA expression in LCM-collected tissue from prostate biopsies. With careful technique and use of appropriate controls, RNA profiling from archived biopsy material is quite feasible showing high correlation to frozen tissue.

An ultrasonically actuated silicon-microprobe-based testicular tubule assay.

Microdissection testicular sperm extraction (TESE) is an invasive surgical procedure in which sparsely located healthy larger diameter tubules carrying viable spermatazoa are identified by visual examination of the seminiferous tubules of the infertile testis under a microscope, and biopsies of regions of interest are performed. In this paper, we report on microfabricated silicon microprobes integrated with an ultrasonic horn actuator and strain gauges for microdissection probe-TESE (MP-TESE) surgery. The microprobes, with axial-force-sensitive polysilicon strain gauges, have high force sensitivity (-0.4 V/N). The probes were used to detect the boundaries between seminiferous tubules, thus enabling identification of individual tubule diameters. Insertion experiments were performed on rat testis tissue, and by monitoring the tubule puncture in the recorded force, we were able to estimate the average diameter approximately 41.2 +/- 1.6 microm of the sperm-carrying tubules in samples. We have also demonstrated the ability to sense the existence of larger tubules embedded in a mess of thinner tubules, using an analytically calculated expression for the distribution of sizes measured by the microprobe. This information is important in MP-TESE to distinguish tubules with and without fertile sperm, potentially eliminating the large incision currently required for optical spermatazoa localization.

[A simple method for assessment of RNA integrity in laser capture microdissection samples].

OBJECTIVE: To develop a simple method for assessment of RNA integrity in laser capture microdissection (LCM) samples. METHODS: The total RNA were isolated from the LCM samples and the sections before and after microdissection and examined by agarose gel electrophoresis. Real-time PCR was employed to assess the RNA from LCM samples, and the quantity of RNA was theoretically estimated according to the average total RNA product in mammalian cells (10 ng/1000 cells). RESULTS: When the total RNA from the sections before and after microdissection was intact, the RNA from LCM samples also had good quality, and the 28S and 18S rRNAs were visualized by ethidium bromide staining. Real-time PCR also showed good RNA quality in the LCM samples. CONCLUSION: A simple method for quantitative and qualitative assessment of the RNA from LCM samples is established, which can also be applied to assessment of DNA or proteins in LCM samples.

Laser-capture microdissection in prostate cancer research: establishment and validation of a powerful tool for the assessment of tumour-stroma interactions.

OBJECTIVES: To describe our experience with the optimization and validation of laser-capture microdissection (LCM) for biomarker analysis in prostate tissues. As LCM allows the separation of benign and malignant epithelial structures and stromal elements, it not only allows identification of the source of the biomarker, but might also accentuate gene or protein expression changes by reducing contamination by other cellular elements. MATERIALS AND METHODS: In all, 19 fresh-frozen prostate tissue samples were subjected to LCM, with the cDNA being analysed using quantitative polymerase chain reaction for several genes, to identify the optimum number of cells for capture, as well as gene markers assessing for the purity of the captured cells. The localization was further confirmed by in situ hybridization. RESULTS: Prostate-specific antigen (PSA) and cytokeratin 8, were expressed solely by epithelial cells, whereas hepatocyte growth factor (HGF) and tissue inhibitor of metalloproteinases-3 (TIMP3) were expressed only by stromal cells, and the levels of transcripts of these genes were unaltered between benign and malignant tissues. CONCLUSIONS: These data suggest that PSA, cytokeratin 8, HGF and TIMP3 are reliable gene markers of purity of epithelial and stromal compartments for LCM of prostate tumours. Although this technique is not new and is increasingly used in laboratories, it needs optimization and stringent validation criteria before data analysis. This applies to all tissue types subjected to LCM.

Assessment of normalization strategies for quantitative RT-PCR using microdissected tissue samples.

Gene expression measurement techniques such as quantitative reverse transcriptase (qRT)-PCR require a normalization strategy to allow meaningful comparisons across biological samples. Typically, this is accomplished through the use of an endogenous housekeeping gene that is presumed to show stable expression levels in the samples under study. There is concern regarding how precisely specific genes can be measured in limited amounts of mRNA such as those from microdissected (MD) tissues. To address this issue, we evaluated three different approaches for qRT-PCR normalization of dissected samples; cell count during microdissection, total RNA measurement, and endogenous control genes. The data indicate that both cell count and total RNA are useful in calibrating input amounts at the outset of a study, but do not provide enough precision to serve as normalization standards. However, endogenous control genes can accurately determine the relative abundance of a target gene relative to the entire cellular transcriptome. Taken together, these results suggest that precise gene expression measurements can be made from MD samples if the appropriate normalization strategy is employed.

Preparation and quality assessment of RNA from cell-specific samples obtained by laser microdissection.

Laser microdissection is a powerful tool to obtain cell-specific isolates from complex tissue samples. This chapter outlines how to prepare plant material for microdissection and methods to extract and measure high-quality RNA suitable for a variety of different downstream applications.

Influence of new technologies on thyroid surgery: state of the art.

The history of thyroid surgery starts with Billroth, Kocher and Halsted, who developed the technique for thyroidectomy between 1873 and 1910. In general, the essential objectives for thyroidectomy are conservation of the parathyroid glands, avoidance of injury to the recurrent laryngeal nerve, an accurate hemostasis and an excellent cosmesis. In the last 20 years, major improvements and new technologies have been proposed and applied in thyroid surgery; among these are mini-invasive thyroidectomy, new devices for achieving hemostasis and dissection, regional anesthesia and intraoperative neuro-monitoring.

Assessment of RET/PTC oncogene activation in thyroid nodules utilizing laser microdissection followed by nested RT-PCR.

Single palpable nodules of the thyroid gland are common in clinical practice; the majority of such lesions are benign. However, noninvasive thyroid nodules that exhibit borderline morphological signs of papillary cancer represent a diagnostic challenge. Rearrangements of the RET oncogene have been proposed as a marker for papillary thyroid cancer. In this chapter, methods for the analysis of the RET oncogene in laser microdissected papillary thyroid cancer tissue are described.