Laser Capture Microdissection Method Research Articles

Abstract 11703: Proteomic Analysis for the Formation of the Plexiform Lesions of a Rat Pulmonary Arterial Hypertension Model

Introduction: Although plexiform lesions (PLs) are the hallmark of the severe pulmonary arterial hypertension, the mechanism of this lesion formation has not been fully elucidated. In this study, to explore the pathways associated PLs formation, we examined the proteome analysis using vascular tissue before and after the formation of PLs obtained by laser capture microdissection (LCM). Methods: 7 weeks old rats were injected Sugen 5416 subcutaneously, followed by exposed to hypoxia for 3 weeks. In addition, the rats were kept in room air for 5 weeks (8-week model) or 10 weeks (13-week model) thereafter. We obtained the tissue of vessels with medial hypertrophy from 8-week models (n=5) and PLs from 13-week models (n=5) by LCM. Those micro-dissected samples were then subject to a qualitative and then quantitative proteomics approach. Differentially expressed proteins (DEPs) were defined by abundance in mass spectrometry (MS) (P-value < 0.05 and |log2 (fold change)| > 0.5 or proteins deteced only in lesions extracted from one model). In addition, we investigated enhanced pathway by gene ontology (GO) analysis of the identified DEPs in each of the 8-week and 13-week models. Results: We identified 718 uniquely expressed proteins including 57 DEPs (31 in 8-week model versus 26 in 13-week model) by using LCM-MS. GO analysis revealed that the actin cytoskeleton organization, actin filament-based process, and actomyosin structure organization processes were enhanced in 8-week model. On the other hand, the innate immune system, G2/M check points, and cellular response to reactive oxygen species were enhanced in 13-week model. Conclusion: Pulmonary vascular lesions of 8-week and PLs of 13-week Sugen hypoxia model evaluated by proteomics using samples obtained by LCM methods showed differential protein expression. Analysis of these DEPs may contribute to elucidate the mechanisms of PLs formation.

Epigenetically altered macrophages promote development of diabetes-associated atherosclerosis.

Atherosclerosis (AS) risk is elevated in diabetic patients, but the underlying mechanism such as involvement of epigenetic control of foam macrophages remains unclear. We have previously shown the importance of immune regulation on endothelial cells to AS development in diabetes. In this study, we examined the hypothesis that diabetes may promote AS through modification of the epigenetic status of macrophages. We employed the Laser Capture Microdissection (LCM) method to evaluate the expression levels of key epigenetic regulators in both endothelial cells and macrophages at the AS lesions of patients. We then assessed the correlation between the significantly altered epigenetic regulator and serum levels of low-density Lipoprotein (LDL), triglycerides (TRIG) and high-density Lipoprotein (HDL) in patients. In vitro, the effects of high glucose on glucose utilization, lactate production, succinate levels, oxygen consumption and polarization in either undifferentiated or differentiated bone marrow-derived macrophages (BMDMs) were analyzed. The effects of depleting this significantly altered epigenetic regulator in macrophages on AS development were assessed in AS-prone diabetic mice. Histone deacetylase 3 (HDAC3) was identified as the most significantly altered epigenetic regulator in macrophages from the AS lesions in human diabetic patients. The levels of HDAC3 positively correlated with high serum LDL and TRIG, as well as low serum HDL. High glucose significantly increased glucose utilization, lactate production, succinate levels and oxygen consumption in cultured macrophages, and induced proinflammatory M1-like polarization. Macrophage depletion of HDAC3 significantly attenuated AS severity in AS-prone diabetic mice. Epigenetically altered macrophages promote development of diabetes-associated AS, which could be prevented through HDAC3 depletion.

Laser Capture Microdissection Optimization for High-Quality RNA in Mouse Brain Tissue.

Laser Capture Microdissection (LCM) is a method that allows one to select and dissect well-defined structures, specific cell subpopulations, or even single cells from different types of tissue for subsequent extraction of DNA, RNA, or proteins. Its precision allows the dissection of specific groups of cells, avoiding unwanted cells. However, despite its efficiency, several steps can affect the sample RNA integrity. RNA instability represents a challenge in the LCM method, and low RNA integrity can introduce biases, as different transcripts often have different degradation rates. Here we describe an optimized protocol to provide good-concentration and high-quality RNA from specific structures: dentate gyrus and CA1 in the hippocampus, basolateral amygdala, and anterior cingulate cortex of mouse brain tissue. However, the protocol is applicable to other areas of interest. © 2022 Wiley Periodicals LLC. Basic Protocol: Laser capture microdissection of mouse brain tissue.

The use of combined PCR, fluorescence in situ hybridisation and immunohistochemical staining to diagnose mucormycosis from formalin-fixed paraffin-embedded tissues.

To develop a comprehensive diagnostic system for mucormycosis from formalin-fixed paraffin-embedded tissues, consisting of own-designed real-time polymerase chain reaction (PCR) assays, fluorescence in situ hybridisation, and immunohistochemical staining. We designed 11 primers and probes for specific real-time PCR assays based on genome sequences, and validated the specificity by Aspergillus, Fusarium, Scedosporium, Lomentospora, Cryptococcus and Candida species. Formalin-fixed paraffin-embedded (FFPE) tissues from forty-four mouse model infected by above fungi were collected and extracted DNA by laser capture microdissection (LCM) and direct extraction methods for real-time PCR assays. In addition, seventeen clinical specimens histopathologically proven for mucormycosis were included for specific detection with the new diagnostic system. The real-time PCR assays allowed detection of a minimum of 10CFU/ml equivalent gDNA of each species. No cross-reaction with gDNA among species was noted. From mouse model specimens, the sensitivity of real-time PCR in samples extracted with LCM versus direct extraction method was 100% versus 91.43% at Mucorales level and 80% versus 45.71% at species level, respectively. The specificity was 100%. From clinical samples, LCM combined with real-time PCR can test 88.24% (15/17) of Mucorales. Sensitivities of fluorescence in situ hybridisation (FISH) and immunohistochemical staining (IHC) were 70.59% and 41.18%, respectively. Combined LCM-RT-PCR, FISH and IHC yielded positive results in all samples. The combination diagnostic system we developed is a culture-independent and robust method which enables rapid species identification from FFPE tissues for timely diagnosis of mucormycosis.

Determination of the virulence of single nucleopolyhedrovirus occlusion bodies using a novel laser capture microdissection method.

Determination of the virulence of occlusion bodies (OBs), which are the horizontal transmission structures of nucleopolyhedroviruses (NPVs), is an important area of baculovirology. A method for inoculating an insect with an isolated OB was developed using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) infection of second instar Helicoverpa armigera larvae as a model NPV-host pathosystem. In this novel method, laser capture microdissection (LCM) was used to directly catapult single OBs onto the surface of insect diet in bioassay containers. Since exposure via the natural oral horizontal transmission route of each larva to a single OB was established and not subject to chance variation, the method facilitated determination of the insect mortality rate (4.8%) associated with exposure to single HearNPV OBs. Droplet feeding bioassays confirmed that the novel method did not reduce OB virulence. The LCM method sets a foundation for virulence and genetic diversity studies based on single NPV OBs.

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants.

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Early Enhanced Leucine-Rich α-2-Glycoprotein-1 Expression in Glomerular Endothelial Cells of Type 2 Diabetic Nephropathy Model Mice

Abnormal angiogenesis plays a major role in the development of early stage diabetic nephropathy. Vascular endothelial growth factor (VEGF) is a classical proangiogenic factor that regulates abnormal glomerular angiogenesis linked to glomerular hypertrophy in the early stage of diabetic nephropathy. Leucine-rich α-2-glycoprotein-1 (LRG1) was recently reported as a novel proangiogenic factor that is expressed in endothelial cells and promotes angiogenesis by modulating the transforming growth factor-β signaling pathway. However, the pathophysiology of LRG1 in diabetic nephropathy remains largely unknown. In the present study, we investigated intrarenal expression of the novel proangiogenic factor LRG1 in diabetic db/db mice by immunohistochemistry and a laser capture microdissection method during the development of diabetic nephropathy. We hypothesized that glomerular LRG1 expression is increased earlier than VEGF expression under conditions of pathological angiogenesis in the early stage of diabetic nephropathy. Thus, we compared glomerular expression of VEGF and LRG1 in diabetic db/db mice at 16 and 24 weeks of age. At 16 weeks, diabetic db/db mice exhibited glomerular hypertrophy with abnormal angiogenesis characterized by endothelial cell proliferation, which was concomitant with an increase in LRG1 expression of glomerular endothelial cells. However, glomerular VEGF expression was not increased at this early stage. At 24 weeks, the features of early diabetic nephropathy in db/db mice had developed further, along with further enhanced glomerular LRG1 expression. At this late stage, glomerular VEGF and fibrosis-related-gene expression was also significantly increased compared with nondiabetic db/m mice. These results suggest that LRG1 plays a pivotal role in the initial development of diabetic nephropathy by promoting abnormal angiogenesis, thereby suggesting that LRG1 is a potential preemptive therapeutic target of diabetic nephropathy.

Label-Free LC-MS/MS Strategy for Comprehensive Proteomic Profiling of Human Islets Collected Using Laser Capture Microdissection from Frozen Pancreata.

Diabetes mellitus is caused by either loss of pancreatic islets β-cells (Type 1 Diabetes, T1D), insufficient insulin release in the islet β-cells coupled with insulin resistance in target tissues (Type 2 Diabetes, T2D), or impaired insulin release (genetic forms of diabetes and, possibly, T1D subtypes). The investigation of the islet proteome could elucidate facets of the pathogenesis of diabetes. Enzymatically isolated and cultured (EIC) islets are frequently used to investigate biochemical signaling pathways that could trigger β-cell changes and death in diabetes. However, they cannot fully reflect the natural protein composition and disease process of in vivo islets due to the stress from isolation procedures and in vitro culture. The laser capture microdissection method employs a high-energy laser source to separate the desired cells from the remaining tissue section in an environment which is well conserved and close to the natural condition. Here, we describe a label-free proteomic workflow of laser capture microdissected (LCM) human islets from fresh-frozen pancreas sections of cadaveric donors to obtain an accurate and unbiased profile of the pancreatic islet proteome. The workflow includes preparation of frozen tissue section, staining and dehydration, LCM islets collection, islet protein digestion, label-free Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), database search, and statistical analysis.

Effects of nerve growth factor neutralization on TRP channel expression in laser-captured bladder afferent neurons in mice with spinal cord injury

Nerve growth factor (NGF) is reportedly involved in the changes in C-fiber bladder afferent pathways that induce detrusor overactivity (DO) following spinal cord injury (SCI). This study examined the roles of NGF in TRP channel expression in bladder afferent neurons in mice with SCI using laser-capture microdissection (LCM) methods. Spinal intact (SI) and SCI mice were divided into 3 groups: (1) SI with vehicle treatment; (2) SCI with vehicle treatment; and (3) SCI with anti-NGF antibody. Two weeks after SCI, an osmotic pump was placed subcutaneously into the back of the mice and vehicle or anti-NGF antibody was administered at a rate of 10 μg/kg per hour for two weeks. Four weeks after SCI, the L6 dorsal root ganglia (DRG) were removed. Expression of the TRPV1, TRPC1, TRPC3, and TRPC6 genes was analyzed using real-time polymerase chain reaction (PCR) following LCM of the bladder afferent neurons, which were labeled by Fast Blue injected into the bladder wall 1 week prior to tissue removal. The mRNA expression of TRPV1 was found to be higher in vehicle-treated SCI mice than in SI mice. The expression level of TRPC3 and TRPC6 in vehicle-treated SCI mice was lower than in SI mice. However, in SCI mice treated with anti-NGF antibody, the mRNA expression of TRPV1 was lower, and the mRNA levels of TRPC3 and TRPC6 were higher than in vehicle-SCI mice. These results suggest that the NGF-dependent changes in specific TRP channel genes, such as TRPV1, TRPC3, and TRPC6, could be involved in SCI-induced afferent hyperexcitability and DO.

MP45-06 BLADDER UROTHELIAL AND AFFERENT DYSFUNCTIONS UNDERLYING BLADDER OVERACTIVITY IN RATS WITH CHEMICALLY INDUCED PROSTATIC INFLAMMATION

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2018MP45-06 BLADDER UROTHELIAL AND AFFERENT DYSFUNCTIONS UNDERLYING BLADDER OVERACTIVITY IN RATS WITH CHEMICALLY INDUCED PROSTATIC INFLAMMATION Shinsuke Mizoguchi, Amanda S. Wolf-Johnson, Takahisa Suzuki, Eiichiro Takaoka, Donald B. DeFranco, Zhou Wang, Lori A. Birder, and Naoki Yoshimura Shinsuke MizoguchiShinsuke Mizoguchi More articles by this author , Amanda S. Wolf-JohnsonAmanda S. Wolf-Johnson More articles by this author , Takahisa SuzukiTakahisa Suzuki More articles by this author , Eiichiro TakaokaEiichiro Takaoka More articles by this author , Donald B. DeFrancoDonald B. DeFranco More articles by this author , Zhou WangZhou Wang More articles by this author , Lori A. BirderLori A. Birder More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1445AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There is increasing evidence showing the positive correlation between prostatic inflammation and lower urinary tract symptoms (LUTS) in males with benign prostatic hyperplasia (BPH). Therefore, this study used a rat model of non-bacterial prostatic inflammation to investigate the changes in bladder activity and molecular characteristics of bladder afferent pathways and urothelium after prostatic inflammation. METHODS Male SD rats were used, and prostatic inflammation was induced by formalin (5%; 50 µl per lobe) injection into bilateral ventral lobes of the prostate. Bladder function was evaluated 1 and 4 weeks after formalin injection using awake cystometry. Also, after 10 days of prostatic inflammation, prostaglandin E2 (PGE2) concentrations in bladder tissues were measured by ELISA. In bladder mucosa, protein levels of TRPV1 and PGE2 receptors (EP1 to 4) as well as mRNA levels of ASICs (1-3), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were evaluated by Western blot and RT-PCR, respectively. NGF immunoreactivity in bladder sections was also examined. In addition, mRNA levels of TRPV1 and TRPA1 receptors were measured in bladder afferent neurons labeled by Fast Blue (FB) injected into the bladder wall using laser-capture microdissection and RT-PCR methods. RESULTS Compared to vehicle-injected rats, formalin-treated rats exhibited a significant (p<0.05) decrease in intercontraction intervals in cystometry at 1 and 4 weeks after formalin injection. Tissue inflammation evident as inflammatory cell infiltration was found in the prostate, but not in the bladder from formalin-injected rats. In rats with prostatic inflammation (10 days), bladder PGE2 concentrations were significantly increased, and protein levels of TRPV1 and EP4 receptors as well as mRNA levels of all ASICs, BDNF and NGF were significantly elevated in bladder mucosa of formalin-injected rats. NGF immunoreactivity was also increased in bladder urothelium after prostatic inflammation. In FB-labeled bladder afferent neurons, mRNA levels of TRPV1 and TRPA1 were increased compared to non-labelled neurons in prostatic inflammation rats. CONCLUSIONS These results indicate that non-bacterial inflammation confined in the prostate induces bladder overactivity, enhanced PGE2 production in the bladder and upregulations of receptors and growth factors in bladder urothelium and/or afferent pathways, which could contribute to storage LUTS in BPH patients with prostatic inflammation. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e599 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Shinsuke Mizoguchi More articles by this author Amanda S. Wolf-Johnson More articles by this author Takahisa Suzuki More articles by this author Eiichiro Takaoka More articles by this author Donald B. DeFranco More articles by this author Zhou Wang More articles by this author Lori A. Birder More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Laser capture microdissection of pigment cells from Salmonid fish skin to identify differentially expressed genes in an enriched cell population

In order to determine the function of pigment cell types in different pigment pattern of skin, we have applied the laser capture microdissection method (LCM) for isolation of different pigment cell types from skin sections of marble trout (Salmo marmoratus) and brown trout (S. trutta), characterized with labyrinthine and spotted skin pattern, respectively. RNA was isolated from dissected pigment cell populations and used for expression analysis. Expression profiles of candidate genes, potentially involved in pigmentation, in LCM isolated pigment cell population, were more specific compared to differently pigmented skin regions, containing a heterogeneous mixture of cells, including more types of chromatophores. Moreover, they could provide important information about particular pigment cell type function and its contribution to different pigment pattern. Protocol of combined LCM and genomic approach to study single pigment cell populations can be applied in the future, but needs to be further optimized in order to yield RNA of sufficient quantity and quality required for i.e. transcriptomic analyses.

Development of Methods for Selective Gene Expression Profiling in Tertiary Lymphoid Structure Using Laser Capture Microdissection.

Tertiary lymphoid structures (TLS) are de novo lymphoid formations that are induced within tissues during inflammatory episodes. TLS have been reported at various anatomic sites and in many different contexts like cancer, infections, autoimmunity, graft rejection, and idiopathic diseases. These inducible, ectopic, and transient lymphoid structures exhibit the prototypical architecture found within secondary lymphoid organs (SLO) and have been recently appreciated as a major driver of the local adaptive immune reaction. As TLS emerge within tissues, the isolation in situ and the molecular characterization of these structures are challenging to operate. Laser capture microdissection (LCM) is a powerful tool to isolate selective structural components and cells from frozen or paraffin-embedded tissues. We and other groups previously applied LCM to decipher the molecular network within TLS and uncover their intrinsic connection with the local microenvironment. In this chapter, we describe a detailed LCM method for selecting and isolating TLS in situ to perform comprehensive downstream molecular analyses.

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury.

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

Stereotactic Atlas-Guided Laser Capture Microdissection of Brain Regions Affected by Traumatic Injury

The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial distribution. Such techniques also potentially skew gene expression analysis because the process itself can alter expression patterns in individual cells. Here we describe a laser capture microdissection (LCM) method to selectively collect specific brain regions affected by traumatic brain injury (TBI) by using a modified Nissl (cresyl violet) staining protocol and the guidance of a rat brain atlas. LCM provides access to brain regions in their native positions and the ability to use anatomical landmarks for identification of each specific region. To this end, LCM has been used previously to examine brain region specific gene expression in TBI. This protocol allows examination of TBI-induced alterations in gene and microRNA expression in distinct brain areas within the same animal. The principles of this protocol can be amended and applied to a wide range of studies examining genomic expression in other disease and/or animal models.

Periostin expression in cancer-associated fibroblasts of invasive ductal breast carcinoma.

Periostin (POSTN) is a secreted cell adhesion glycoprotein that plays an important role in proliferation, adhesion and migration processes, as well as in regulation of mechanisms related to epithelial-mesenchymal transition (EMT). It also plays a key role in angio- and lymphangiogenesis and in formation of distant metastases. The aim of this work was to determine expression of POSTN in invasive ductal breast carcinoma (IDC) and in non-invasive ductal carcinoma insitu (DCIS) and to correlate its expression with clinicopathological parameters. Material for immunohistochemical studies (IHC) comprise of 70IDC cases, 44 DCIS cases and 21cases of fibrocystic change (FC). Frozen (-80˚C) fragments of tumours taken from 41patients with IDC were used for molecular studies (real-time PCR), including 11 cases of IDC subjected to laser capture microdissection (LCM). POSTN expression was shown mainly in tumour stromal cells, i.e. cancer-associated fibroblasts (CAFs). Statistically significant higher level of POSTN expression in CAFs in IDC as compared to FC (p<0.0001) was observed. Additionally, statistically elevated expression level of POSTN in CAFs in IDC relative to DCIS (p<0.0001) and significantly increased expression of POSTN in CAFs in DCIS in comparison to FC (p=0.0158) was also shown. High level of POSTN expression in CAFs in IDC (>8 IRS points) was significantly correlated with tumour malignancy grade (G) (p=0.0070). Moreover, higher POSTN expression by CAFs was associated with patient shorter overall survival. Significant increase of POSTN expression on mRNA and protein level in CAFs in IDC with the growing malignancy grade of the tumours (G) was shown. Furthermore, with the use of LCM method, statistically significant higher expression of mRNA POSTN in stromal cells relative to cancer cells (p<0.001) was noted. POSTN might be a factor playing an important role in the mechanism of IDC progression.

Abstract 2301: The generation of colorectal cancer mouse model based on microsatellite instability and the identification of transforming growth factor-beta signal target

Abstract Background &amp; Aims: The transforming growth factor-beta (TGF-beta) signal is a tumor-suppressor pathway that is commonly inactivated in about 90% of microsatellite instability (MSI) colorectal cancer (CRC). However, there was little evidence what gene is regulated by TGF-beta signal in the multistep progression sequence of CRC. The first aim of the present study was to generate a mouse model that is null for Tgfbr2 and Apc in the colon epithelium and forms tumors in the colon. The second aim was to analyze the tumors that arose in the mice model for the purpose to identify the gene regulated by TGF-beta signal. Method &amp; Result: Previously we have described the generation of the ‘CDX2P-G19Cre;Apcflox/flox mice’ (called Apc KO mice) which is randomly null for Apc in the colonic epithelium. By mating Tgfbr2flox/flox mice with Apcflox/flox and CDX2P9.5-G19Cre mice, we have finally generated a mouse model ‘CDX2P-G19Cre;Apcflox/flox;Tgfbr2flox/flox mice’ (called Apc+Tgfbr2 KO mice) which is null for Apc and Tgfbr2. In these model, the tumors with well differentiated adenocarcinoma arose mainly in proximal colon and most of mice died at 4 weeks age due to tumor bleeding. Therefore the mice were harvested at 3 weeks age to evaluate the development of colon tumors. Total RNAs of only cancerous tissue areas were extracted from frozen samples by the laser capture microdissection method. We compared gene expression profiles of these mice's tumors (n = 3, respectively) with Mouse Exon 1.0 ST Array (Affymetrix). Gene X expression of Apc+Tgfbr2 KO mice tumors was most highly upregurated by 9.25-fold compared with Apc KO mice (p = 0.045). The array data was validated by quantitative PCR. For human CRC samples, mutations of repetitive mononucleotide tracts in the coding regions of TGFBR2 were identified by direct sequencing. By immunohistochemical analysis, the expression of X was classified according to the percentage of stained cancer cells. The expression was considered to be ‘positive’ if ≥30% of cancer cells were stained. An analysis demonstrated that 11 (100%) of 11 mutated TGFBR2 cases were positive for X, whereas 10 (66.7%) of 15 wild type TGFBR2 cases were positive (P = 0.033), indicating that high expression of X was correlated with TGFBR2 mutation in human CRCs samples. Additionally, the cell proliferation assay revealed that silencing of X led to a significant reduction in CRC cell proliferation. Conversely, forced expression of X enhanced CRC cell proliferation in vitro. Conclusion: We have generated an in vivo model system that Apc and Tgfbr2 were inactivated only in the colonic epithelium and tumors with well differentiated adenocarcinoma arose mainly in proximal colon. The analysis of this model revealed that Gene X is regulated by a TGF-beta signal and likely promotes cell proliferation in CRC. Citation Format: Masashi Miguchi, Takao Hinoi, Manabu Shimomura, Tomohiro Adachi, Yasufumi Saito, Hiroaki Niitsu, Masatoshi Kochi, Yusuke Sotomaru, Hideaki Ijichi, Tsuneo Ikenoue, Kunitoshi Shigeyasu, Kohji Tanakaya, Kazuhiro Sentani, Naohide Oue, Wataru Yasui, Hideki Ohdan. The generation of colorectal cancer mouse model based on microsatellite instability and the identification of transforming growth factor-beta signal target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2301. doi:10.1158/1538-7445.AM2015-2301

Editorial: The Claustrum: charting a way forward for the brain's most mysterious nucleus.

Editorial: The Claustrum: charting a way forward for the brain's most mysterious nucleus.

Phloem-exudate proteome analysis of response to insect brown plant-hopper in rice

Brown plant-hopper (Nilaparvata lugens Stål, BPH), one of the most devastating agricultural insect pests of rice throughout Asia, ingests nutrients from rice sieve tubes and causes a dramatic yield loss. Planting resistant variety is an efficient and economical way to control this pest. Understanding the mechanisms of host resistance is extremely valuable for molecular design of resistant rice variety. Here, we used an iTRAQ-based quantitative proteomics approach to perform analysis of protein expression profiles in the phloem exudates of BPH-resistant and susceptible rice plants following BPH infestation. A total of 238 proteins were identified, most of which were previously described to be present in the phloem of rice and other plants. The expression of genes for selected proteins was confirmed using a laser capture micro-dissection method and RT-PCR. The mRNAs for three proteins, RGAP, TCTP, and TRXH, were further analyzed by using in situ mRNA hybridization and localized in the phloem cells. Our results showed that BPH feeding induced significant changes in the abundance of proteins in phloem sap of rice involved in multiple pathways, including defense signal transduction, redox regulation, and carbohydrate and protein metabolism, as well as cell structural proteins. The results presented provide new insights into rice resistance mechanisms and should facilitate the breeding of novel elite BPH-resistant rice varieties.

MP25-05 ENHANCED BLADDER PAIN SENSITIVITY DUE TO THE PROSTATE-TO-BLADDER AFFERENT CROSS SENSITIZATION IN A RAT MODEL OF PROSTATITIS

You have accessJournal of UrologyInfections/Inflammation of the Genitourinary Tract: Prostate & Genitalia1 Apr 2015MP25-05 ENHANCED BLADDER PAIN SENSITIVITY DUE TO THE PROSTATE-TO-BLADDER AFFERENT CROSS SENSITIZATION IN A RAT MODEL OF PROSTATITIS Tsuyoshi Majima, Yasuhito Funahashi, Katsumi Kadekawa, Momokazu Gotoh, and Naoki Yoshimura Tsuyoshi MajimaTsuyoshi Majima More articles by this author , Yasuhito FunahashiYasuhito Funahashi More articles by this author , Katsumi KadekawaKatsumi Kadekawa More articles by this author , Momokazu GotohMomokazu Gotoh More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.1208AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In chronic pelvic pain syndrome/chronic prostatitis (CPPS/CP), patients suffer from pelvic pain derived not only from the prostate, but also from the lower urinary tract (LUT). However, the mechanisms remain unclear; therefore, we investigated whether prostatic inflammation induced enhanced pain sensitivity in the LUT and bladder overactivity using a rat model of prostatitis. METHODS Adult Male SD rats were divided into two groups; control and prostatitis groups. (1) Formalin was injected into the prostate in the prostatitis group, while saline was injected in the control group. The rats were placed in metabolic cages to evaluate the voiding behavior 1 week after injection. Cystometry was also performed 1 week after injection to measure intercontraction intervals (ICI) of the voiding reflex. (2) Resiniferatoxin (RTx) was instilled into the bladder to evaluate nociceptive behavior such as licking behavior (urethral pain) and freezing behavior (bladder pain) at 1 week after injection. (3) Organ-specific afferent neurons were labelled by retrograde axonal transport of fluorescent dyes, Fast Blue (FB) or DiI, injected into the bladder wall or the prostate, respectively. Dye-labelled afferent neurons in S1 DRG were dissected using laser-capture microdissection method, and mRNA levels of TRP and ATP receptors were measured by real-time RT-PCR. RESULTS 1) In the metabolic cage study, the average micturition volume was decreased significantly in prostatitis rats (n=6) (0.46 ± 0.08 to 0.33 ± 0.07 mL, p = 0.005) without changes in control rats (n=6)(0.42 ± 0.03 to 0.41 ± 0.03 mL). In cystometry, the average ICI was significantly shorter in prostatitis rats (n=7) than in control (n=6) (791 ± 139 vs 1175 ± 146 sec, p = 0.026). (2) There were no significant differences in licking events between two groups (n=6, each group). However, the number of freezing events during 10-15mins after RTx administration into the bladder was significantly (p=0.01) increased in the prostatitis group than in the control group (20±1 and 11±2, respectively). (3) TRPV1, TRPA1 and P2X2 mRNA were increased in not only prostate-innervating DiI-labeled neurons, but also double-labeled neurons and FB-labeled bladder neurons from prostatitis rats compared to control rats CONCLUSIONS These results indicate that prostatic inflammation enhances pain sensitivity of the bladder and induces bladder overactivity in rats, as often seen in patients with CPPS/CP. Also, the prostate-to-bladder afferent cross-sensitization could be a mechanism underlying pelvic pain symptoms in CPPS/CP. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e286 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tsuyoshi Majima More articles by this author Yasuhito Funahashi More articles by this author Katsumi Kadekawa More articles by this author Momokazu Gotoh More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Laser-capture microdissection for analysis of cell type-specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide-induced cystitis.

This study examined whether the laser-capture microdissection (LCM) method can achieve separation of urothelial cells from detrusor cells or superficial urothelial cells from intermediate/basal urothelial cells, using α-smooth muscle actin (SMA) and cytokeratin 20 (CK20). In addition, we investigated the changes in expression of muscarinic receptors in laser-captured urothelial and detrusor cells in rats with chronic cystitis. Female SD rats were injected with cyclophosphamide (75 mg/kg) intraperitoneally at day 1, 4, 7 and 10 to induce chronic cystitis. Saline was injected in the same protocol for controls. Bladder specimens were cut at 8 μm thickness, fixed in 70% ethanol and lightly stained by hematoxylin and eosin, and then superficial urothelium, intermediate/basal urothelium and detrusor muscles were laser-captured separately. Real-time PCR was performed to examine expressions of α-SMA, CK20, muscarinic 2 receptors (M2R) and muscarinic 3 receptors (M3R). The expression of α-SMA mRNA in detrusor muscle cells was 200 times higher than that in urothelial cells in controls. CK20 mRNA expression in apical urothelial cells was 55 times more than that in detrusor muscle and four times more than that in intermediate/basal urothelial cells. Expressions of M2R and M3R mRNA were increased in urothelial cells and decreased in detrusor muscles following chronic cystitis. The LCM could be useful for tissue collection of detrusor muscle and different layers of urothelial cells with minimal contamination of other cell types, and cell type-specific changes in molecular expression could accurately be analyzed. Increased expression of urothelial MR might enhance urothelial-afferent interactions to induce bladder overactivity/pain conditions associated with bladder inflammation.