Histological Protocol Research Articles

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1–7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.

Histochemical Evaluation of Sexual maturation Phases in Solea solea Wild Marine Populations

The histological analyzes performed in gonad cells to verify the occurrence of changes in their anatomy is of crucial importance in order to characterize the dynamics of the gonads during the sexual maturation phase of its cells in specimens of wild marine populations. These histological data, together with other reproductive data, can be used in the monitoring of marine environments in which the Solea solea (Ishiba et al., 2008) lives, namely, the coastal waters of the Iberian Peninsula, Africa and Mediterranean sea (Ishiba et al., 2008). Therefore, the main objective of this study was to evaluate the several cellular types and its quantities and anatomies (Ishiba et al., 2008). The study was performed using a total of 19 specimens of the species Solea solea, collected between the months of January and March of 2011 in two different locations of the Portuguese coast (Aveiro and Leixões, Portugal). The gonad samples were fixated in Formalin-acetic acid-alcohol (FAA) and processed in accordance to the standard histology protocol (Culling et al., 1985). The slices of the gonads were colored with Haematoxylin and Eosin (H&E) (used in the study of the general structure of the gonads, as well as in the identification and localization of the different cellular types of the gonad epithelium). As a result of the study, we found 7 different types of gonad cells: I oogonia (It is not photographed ); II a Chromatin nucleolus oocytes (Fig.1. A); II b perinucleolar oocytes (Fig.1. B); III cortical alveoli oocytes (Fig.1. C); IV vitellogenic oocytes (Fig.1. D); V maturing oocyte (Fig.1. E); VI ovulated oocyte (Fig.1. F), (Hibiya, et al., 1995). The different cellular phases were found in all the gonad samples analyzed in this study and the results corresponded to those expected: a decrease in the size in the cells of the first cellular type, the oogonia and an increase in the size in the cells of all other cellular types found in the study. Histological and histochemical analyses of the Solea solea can be used in anatomic and pathological evaluations of the physiological condition of the animals, as stated previously for the muscles, where histological changes could be observed (such as: degeneration and atrophy of the muscle bundles). In the future, the analytical determination of histological and histochemical changes in the gonads of Solea solea, in conjunction with other analytical processes, such as in situ analyzes, can potentially be used to determine and accurately characterize the physiological condition of animals sampled in any natural environment.

Optical coherence tomography for biofilm detection in chronic rhinosinusitis with nasal polyposis

Chronic rhinosinusitis with nasal polyposis (CRSwNP) is a multifactorial disease that seems to be associated with the presence of microbial biofilms and corresponding subepithelial inflammatory reactions. Optical coherence tomography (OCT) might be applied to detect bacterial and fungal biofilms in patients with CRSwNP. A total of 27 patients with CRSwNP undergoing endoscopic sinus surgery (ESS) were analyzed. The negative control group consisted of six patients undergoing septoplasty for nasal obstruction without CRSwNP. The nasal polyps and inferior turbinate mucosa specimens applied as negative controls were processed to OCT analysis and H.E. and Gram staining. Biofilm was detected in 22 of 27 patients (81.5 %) with CRSwNP and in none of six negative controls. In our series, OCT scan showed an obvious association with the findings of H.E. and Gram staining and was allocated to be a good predictor of biofilm existence. On OCT images, biofilms were displayed as distinct superficial layers with high optical density. It was found that microscopic architecture of biofilms was strongly associated with the integrity of nasal mucosa and to the cellular pattern of subepithelial inflammatory reaction. This study confirmed the presence of microbial biofilms in patients with CRSwNP according to OCT scans and histological analysis. Since biofilms may affect the severity and recurrence rate of CRS treated by ESS they should be detected preoperatively. In conclusion, single application of OCT analysis or combination with conventional histological protocols provides a robust and reliable method for the detection of bacterial and fungal biofilms in CRSwNP. Level of evidence 3b, individual case-control study.

Detection and differentiation of inflammatory versus fibromatous Crohnʼs disease strictures: Prospective comparison of 18F-FDG-PET/CT, MR-enteroclysis, and transabdominal ultrasound versus endoscopic/histologic evaluation

Differentiation between inflammatory and fibromatous strictures in Crohn's disease (CD) is difficult but crucial for therapeutic decisions. The aim of this study was to assess the best noninvasive imaging method for the detection and differentiation of inflammatory and fibromatous stenoses in CD in comparison to endoscopic and histologic evaluation. Patients with suspected CD strictures were included. According to a formalized endoscopic and histologic protocol, strictures were classified as inflammatory, mixed, and fibrostenotic. Strictures were further analyzed using fluorine 18-labeled fluoro-2-deoxy-D-glucose ((18) FDG) / positron emission tomography (PET) low-dose computed tomography (CT), magnetic resonance (MR) enteroclysis and transabdominal ultrasound using standardized scoring systems. Thirty patients with 37 strictures were evaluated (inflamed n = 22; mixed n = 12, fibromatous n = 3). (18) FDG-PET/CT detected 81%, MR-enteroclysis 81%, and ultrasound 68% of the strictures. Correct differentiation rates of strictures were 57% for MRE, 53% for (18) FDG-PET/CT, and 40% for ultrasound. Differences of detection rates and differentiation rates were not statistically significant. When combining transabdominal ultrasound with (18) FDG-PET/CT or MR-enteroclysis all strictures that required invasive treatment were detected. Detection rates of the strictures were not significantly different between (18) FDG-PET/CT, MR-enteroclysis, and ultrasound. Despite good stricture detection rates relating to our gold standard, (18) FDG-PET/CT nor MR-enteroclysis nor ultrasound can accurately differentiate inflamed from fibrotic strictures. A combination of MR-enteroclysis and ultrasound as well as a combination of (18) FDG-PET/CT and ultrasound resulted in a 100% detection rate of strictures requiring surgery or endoscopic dilation therapy, suggesting the combination of these methods as an alternative to endoscopy at least in the group of patients not able to perform an adequate bowel preparation.

Prognostic significance of tumor-infiltrating T cells in ovarian cancer: A meta-analysis

ObjectiveThe presence of T cells within the epithelial component of tumors, as histologic evidence of anti-tumor immunity, has been associated with a survival advantage in multiple studies across diverse patient cohorts. We performed a meta-analysis of studies evaluating the prognostic value of tumor-infiltrating lymphocytes (TIL) on survival among women with ovarian cancer and to investigate factors associated with variations in this effect, including patient characteristics, surgical outcomes, tumor histology, and study protocols. MethodPublished studies that evaluated the association between TIL and patient survival were identified. Descriptive statistics, outcome data, and study quality were extracted from studies that met inclusion criteria. Hazard ratios and 95% confidence intervals were pooled across studies using the random-effects model. Publication bias was investigated using a funnel plot and heterogeneity was assessed with subgroup analysis and I2 statistics. ResultsTen suitable studies comprising 1815 patients with ovarian cancer were analyzed. Our results demonstrate that a lack of intraepithelial TILs is significantly associated with a worse survival among patients (pooled HR: 2.24, 95% CI; 1.71–2.91). Variations in the prognostic value of TIL status based on debulking status, scoring method, and geographic regions were identified. ConclusionsIntraepithelial TILs are a robust predictor of outcome in ovarian cancer and define a specific class of patients, whose distinct tumor biology should be taken into account in devising appropriate therapeutic strategies.

Treatment influencing down-staging in EORTC Melanoma Group sentinel node histological protocol compared with complete step-sectioning: A national multicentre study

AimMetastasis size in melanoma sentinel lymph nodes (SLNs) is an emerging prognostic factor. Two European melanoma treatment trials include SLN metastasis diameters as inclusion criteria. Whilst diameter estimates are sensitive to the number of sections examined, the level of this bias is largely unknown. We performed a prospective multicentre study to compare the European Organisation for Research and Treatment of Cancer (EORTC) recommended protocol with a protocol of complete step-sectioning. MethodsOne hundred and thirty-three consecutive SLNs from seven SLN centres were analysed by five central sections 50μm apart (EORTC Protocol) followed by complete 250μm step-sectioning. ResultsOverall, 29 patients (21.8%) were SLN-positive. The EORTC Protocol missed eight of these metastases (28%), one metastasis measuring less than 0.1mm in diameter, seven measuring between 0.1 and 1mm. Complete step-sectioning at 250μm intervals (Extensive Protocol) missed one metastasis (3%) that measured less than 0.1mm. Thirteen treatment courses (34%) performed if inclusion was based on the Combined Protocol would not be performed if assessed by the EORTC Protocol. Thus, 10 patients would be without completion lymph node dissection (EORTC MINITUB study), whilst three patients would not be eligible for anti-CTLA4 trial (EORTC protocol 18071). The corresponding number with the Extensive Protocol would be three; one patient for the MINITUB registration study and two patients for the anti-CTLA4 study. ConclusionsExamining SLNs by close central sectioning alone (EORTC Protocol) misses a substantial number of metastases and underestimates the maximum metastasis diameter, leading to important changes in patient eligibility for various treatment protocols.

Determination of cardiac histologic injury in a transgenic mouse model of heart failure expressing a novel mitochondrial progesterone receptor (PR-M)

1. To develop an automated histological fibrosis quantification protocol. 2. To determine cardiac histologic injury in a transgenic mouse model of heart failure expressing a human mitochondrial progesterone receptor (PR-M). Transgenic mouse study. PR-M is mitochondrial progesterone receptor isoform that increases cellular respiration. An inducible (TET-On) transgenic mouse model expressing human PR-M under the control of the cardiac myosin heavy chain 6 promoter was developed. PR-M expression was induced with oral doxycycline followed in 2 wks by surgical constant thoracic aortic constriction (cTAC) to increase after-load. Intact male and ovariectomized female mice were treated with progesterone in oil (2.5 mg/day SQ) starting 1 wk prior to cTAC. Mice were sacrificed 8 wks after cTAC and cardiac PR-M expression determined with realtime RT-PCR. Cardiac fibrosis and capillaries were identified with Masson's trichrome and immunostaining with lectin respectively, by an investigator blinded to gene expression and treatment. Histologic images were acquired via digital slide scanning. Morphometrics were computed with ImageJ64 (NIH) and Photoshop CS5 (Adobe Systems). Data from the histologic quantification were analyzed with unpaired Student's t-test. Progesterone treated PR-M positive mice (N = 9) showed greater capillary density (P=0.04), greater capillary density to cardiomyocyte width ratio (P=0.02) and decreased cardiac fibrosis (P=0.03) after cTAC compared to progesterone treated PR-M negative mice (N = 17). The mean myocyte width was not different between the 2 groups (P=0.33). A novel computerized fibrosis quantification method for cardiac tissue was developed. Compared to controls, progesterone treated transgenic mice expressing a human mitochondrial progesterone receptor (PR-M) had fewer histologic signs of cardiac injury. These observations support a role for progesterone via a mitochondrial progesterone receptor in cardiac function.

Conditional Inactivation of the CXCR4 Receptor in Osteoprecursors Reduces Postnatal Bone Formation Due to Impaired Osteoblast Development

Cysteine (C)-X-C motif chemokine receptor 4 (CXCR4), the primary receptor for stromal cell-derived factor-1 (SDF-1), is involved in bone morphogenic protein 2 (BMP2)-induced osteogenic differentiation of mesenchymal progenitors. To target the in vivo function of CXCR4 in bone and explore the underlying mechanisms, we conditionally inactivated CXCR4 in osteoprecursors by crossing osterix (Osx)-Cre mice with floxed CXCR4 (CXCR4(fl/fl)) mice to generate knock-outs with CXCR4 deletion driven by the Osx promoter (Osx::CXCR4(fl/fl)). The Cre-mediated excision of CXCR4 occurred exclusively in bone of Osx::CXCR4(fl/fl) mice. When compared with littermate controls, Osx::CXCR4(fl/fl) mice developed smaller osteopenic skeletons as evidenced by reduced trabecular and cortical bone mass, lower bone mineral density, and a slower mineral apposition rate. In addition, Osx::CXCR4(fl/fl) mice displayed chondrocyte disorganization in the epiphyseal growth plate associated with decreased proliferation and collagen matrix syntheses. Moreover, mature osteoblast-related expression of type I collagen α1 and osteocalcin was reduced in bone of Osx::CXCR4(fl/fl) mice versus controls, suggesting that CXCR4 deficiency results in arrested osteoblast progression. Primary cultures for osteoblastic cells derived from Osx::CXCR4(fl/fl) mice also showed decreased proliferation and impaired osteoblast differentiation in response to BMP2 or BMP6 stimulation, and suppressed activation of intracellular BMP receptor-regulated Smads (R-Smads) and Erk1/2 was identified in CXCR4-deficient cells and bone tissues. These findings provide the first in vivo evidence that CXCR4 functions in postnatal bone development by regulating osteoblast development in cooperation with BMP signaling. Thus, CXCR4 acts as an endogenous signaling component necessary for bone formation.

Overuse of training increases mechanoreceptors in supraspinatus tendon of rats SHR

The presence of mechanoreceptors in tendon after overuse activities can be a further step to learn about tendinopathy and overuse. Studies of tendons mechanoreceptors in rats are rare. We studied 12 isogenic spontaneous hypertensive rats (SHR), which underwent an overuse protocol consisting of an hour per daily session of treadmill running at a speed of 17 m/min, 5 times/week for 4 months. Supraspinatus tendons were evaluated with immunohistochemistry using S100 protein antibodies and histological protocol. Supraspinatus tendons at the end of 4 months of overuse protocol had a high number of media mechanoreceptors (4.3) than controls (0.6). The overexpression of S100 protein antibody in overuse activities maybe could represent a adaptative effort to tendon before the tear.

A Digital Atlas of the Guinea Pig Brain. A Comparative Study of qMRI at 3T with Histological Sections.

Event Abstract Back to Event A Digital Atlas of the Guinea Pig Brain. A Comparative Study of qMRI at 3T with Histological Sections. Marie Drottar1* and Hernan Jara2 1 Boston University, Division of Graduate Medical Sciences: BioImaging Program, United States 2 Boston University, Department of Radiology, Medical School, United States PURPOSE: High quality magnetic resonance images of the guinea pig brain have been collected using quantitative MRI scanning techniques. This series of images reveals a set of digital images of the guinea pig brain in sections that are comparable to a set of images obtained with commonly used histological sectioning and staining protocols. These image sets lend themselves to the construction of a freely accessible and user friendly digital atlas that can be labeled with anatomical structures and used as a searchable database. METHOD AND MATERIALS: One healthy adult male guinea pig was sub-lethally anesthetized and perfused with 4% formalin through the heart in accordance with the IACUC approved protocol at the Mass Eye and Ear Infirmary. The entire brain (still within the cranial vault), was post-fixed for 2 hrs in 4% formalin, and then removed into 0.01M phosphate buffered saline for one week. The specimen was then immobilized in a 2% agarose block. Imaging was accomplished using a z3D-mixed-TSE scanning protocol in a 3T clinical MRI scanner (Philips Medical Systems, Cleveland OH). After Scanning was accomplished, the specimen was removed from agarose, dissected from the cranial vault, and frozen sectioned at 80um on a sliding microtome. Alternate sections were stained with a Nissl stain, and an acetylcholinesterase stain. RESULTS: Three sets of anatomical images were converted to jpeg format, and multiple anatomical structures were labeled in order to make comparisons. CONCLUSION: The quantitative magnetic resonance images (qMRI) provide an anatomical image set which is comparable to the pervasive “gold standard” histological data. These image sets lend themselves easily to the construction of digital image sets which will be accessible, searchable and forms a valuable resource to the research community. This pulse sequence and construction format also has the potential to be applied to in vivo applications. Keywords: digital atlasing Conference: 4th INCF Congress of Neuroinformatics, Boston, United States, 4 Sep - 6 Sep, 2011. Presentation Type: Poster Presentation Topic: Digital atlasing Citation: Drottar M and Jara H (2011). A Digital Atlas of the Guinea Pig Brain. A Comparative Study of qMRI at 3T with Histological Sections.. Front. Neuroinform. Conference Abstract: 4th INCF Congress of Neuroinformatics. doi: 10.3389/conf.fninf.2011.08.00083 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 17 Oct 2011; Published Online: 19 Oct 2011. * Correspondence: Dr. Marie Drottar, Boston University, Division of Graduate Medical Sciences: BioImaging Program, Boston, United States, mdrottar@bu.edu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Marie Drottar Hernan Jara Google Marie Drottar Hernan Jara Google Scholar Marie Drottar Hernan Jara PubMed Marie Drottar Hernan Jara Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Morfometria de testículos y celulas germinales de Allobates femoralis (Boulenger 1883) (Dendrobatidae: Anura: Amphibia)

The present study describes the morphological aspects of the testis and presents a general characterization of the seminiferous elements of adult specimens (N=8) of Allobates femoralis (Boulenger, 1883) collected at the Panguana Biological Station (9°37'S 74°56'W), 250 masl, upstream of the Pachitea river, Yuyapichis district, Puerto Inca province, Huanuco, Peru. The amphibians (25.99 ± 0.97 mm in length) were dissected and their testes were isolated for macroscopic descriptions; later, routine histological protocols for microscopic analysis were done, colored with hematoxylin and eosin dye. The germinal stages were described and measured with the help of an ocular micrometer. The biometrics of the testes were: major axis = 2.254 mm (SD=0.282), minor axis = 1.343 mm (SD=0.181), width = 0.971 (SD=0.097). At a microscopic level we found the albuginea coat (6.51 ± 1.74 im width) formed by connective tissue and smooth muscle. The seminiferous tubules (162.56 ± 47.61im) were delimited by a thin layer of connective tissue. Regarding the microscopic features, spermatogenesis in A. femoralis occurs into the seminiferous tubules where the cellular elements are organized in spermatocysts. Each cyst contains cells in the same stage of development. The characterization of each cell type allowed the identification of the germ line cells: 1) Spermatogonia, isolated cells or forming groups of the most voluminous cells of the germinal linage (7.07 ± 1.187 im) with an irregular nucleus shape. They laid very close to the seminiferous tubule. 2) Spermatocyte I, with a smaller form than spermatogonias (6.95 ± 1.21 im). 3) Spermatids; with a round shape, as they advance in their development, they elongated slightly and are organized into bundles whose heads are attached between them. 4) Spermatozoa, elongated with the flagellum lightly stained, are well compacted cells with the nucleus and cytoplasm reduced, elongated and extend into the lumen of the seminiferous tubule. It is reported for the first time the macroscopic and microscopic description of the testis of A. femoralis in Peru.

Reliable magnetic reversible assembly of complex microfluidic devices: fabrication, characterization, and biological validation

Current standard procedures for fabrication of microfluidic devices combine polydimethylsiloxane (PDMS) replica molding with subsequent plasma treatment to obtain an irreversible sealing onto a glass/silicon substrate. However, irreversible sealing introduces several limitations to applications and internal accessibility of such devices as well as to the choice of materials for fabrication. In the present work, we describe and characterize a reliable, flexible and cost effective approach to fabricate devices that reversibly adhere to a substrate by taking advantage of magnetic forces. This is shown by implementing a PDMS/iron micropowder layer aligned onto a microfluidic layer and coupled with a histology glass slide, in union with either temporary or continuous use of a permanent magnet. To better represent the complexity of microfluidic devices, a Y-shaped configuration including lower scale parallel channels on each branch has been employed as reference geometry. To correctly evaluate our system, current sealing methods have been reproduced on the reference geometry. Sealing experiments (pressure control, flow control and hydraulic characterization) have been carried out, showing consistent increases in terms of maximum achievable flow rates and pressures, as compared to devices obtained with other available reversible techniques. Moreover, no differences were detected between cells cultured on our magnetic devices as compared to cells cultured on permanently sealed devices. Disassembly of our devices for analyses allowed to stain cells by hematoxylin and eosin and for F-actin, following traditional histological processes and protocols. In conclusion, we present a method allowing reversible sealing of microfluidic devices characterized by compatibility with: (i) complex fluidic layer configurations, (ii) micrometer sized channels, and (iii) optical transparency in the channel regions for flow visualization and inspection.

Evaluation of implant osseointegration with different regeneration techniques in the treatment of bone defects around implants: an experimental study in a rabbit model

The aim of this study was to evaluate the osseointegration of implants placed in areas with artificially created bone defects, using three bone regeneration techniques. The experimental model was the rabbit femur (16), where bone defects were created and implants were placed. The peri-implant bone defects were filled with a deproteinized bovine bone mineral, NuOss™ (N), NuOss™ combined with plasma rich in growth factors (PRGF) (N+PRGF), NuOss™ covered by an RCM(6) membrane (N+M), or remained unfilled (control group [C]). After 4 and 8 weeks, the animals were euthanized and bone tissue blocks with the implants and the surrounding bone tissue were removed and processed according to a histological protocol for hard tissues on non-decalcified ground sections. The samples were studied by light and electron scanning microscopy, histometric analysis was performed to assess the percentage of bone in direct contact with the implant surface and a statistical analysis of the results was performed. In the samples analyzed 4 weeks after implantation, the percentage of bone tissue in direct contact with the implant surface for the four groups were 57.66±24.39% (N), 58.62±20.37% (N+PRGF), 70.82±20.34 % (N+M) and 33.07±5.49% (C). In the samples with 8 weeks of implantation time, the percentage of bone in direct contact was 63.35±27.69% (N), 58.42±24.77% (N+PRGF), 78.02±15.13% (N+M) and 40.28±27.32% (C). In terms of the percentage of bone contact, groups N and N+M presented statistically significant differences from group C in the 4-week trial test (P<0.05; ANOVA). For the 8-week results, only group N+M showed statistically significant differences when compared with group C (P<0.05; ANOVA). In conclusion, the NuOss™ granules/RCM(6) membrane combination presented a percentage of bone contact with the implant surface statistically greater than in the other groups.

Morphological Patterns Identification in PET+ Aneurysmatic Lesions

The size of abdominal aortic aneurysm (AAA) is a well-known risk factor, predictive for rupture. The employment of Positron Emission Tomography (PET) has recently proven to be helpful in determining which AAA are at imminent risk of rupture. We applied our macroscopic sampling and histological examination protocol to I4 AAA (2 of which PET positive), in order to determine the correlations between metabolic activation of the aneurysmal wall and histomorphological findings. Our aim is to obtain a better understanding of the pathogenesis of AAA rupture and to provide a further contribute in their diagnostic evaluation. We applied our histochemical protocol (Hematoxylin-Eosin (HE), Alcian Blue (pH-I), PAS, Masson Thrichromic and Weigert) on aortic aneurysm specimen and evaluated the proliferative index by immunohistochemistry (Ki-67/MIB-I) in order to detect any relationship between PET negative aortic specimen showed follicular lymphocytic aggregatesm especially located within the advated proliferative index (Ki-67) of the flogistic aggregates in our two groups (PET positive and PET negative). We believe that further IHC investigations with enzymes that are well-known implied in AAA pathogenesis (i.e. MMP-8 and MMP-9) should provide more accurate information in predicting the risk of rupture.

The Nodal Location of Metastases in Melanoma Sentinel Lymph Nodes

The design of melanoma sentinel lymph node (SLN) histologic protocols is based on the premise that most metastases are found in the central parts of the nodes, but the evidence for this belief has never been thoroughly tested. The nodal location of melanoma metastases in 149 prospectively analyzed, completely step sectioned, positive SLNs from 96 patients was examined using 3 theoretical protocols, evaluating respectively: (1) the 3 most central step sections only; (2) the 3 most peripheral step sections only; and (3) 3 step sections evenly distributed throughout the individual SLNs. In addition, the size of the metastases located exclusively outside the 2 regional protocols (ie, 3 central sections, and 3 peripheral sections) were measured and compared with each other. The metastasis detection rates of the central, the peripheral, and the evenly distributed protocols were 77%, 79%, and 78%, respectively. No difference in either the mean volume or the maximum diameter of the metastases located exclusively outside the central and the peripheral protocols was found (volume: 0.036 vs. 0.031 mm and diameter: 0.320 vs. 0.332 mm). In SLNs, melanoma metastases are located throughout the nodes. Metastases located exclusively outside the peripheral or the central protocol are equally sized. Complete step sectioning of all SLNs will ensure both high metastasis detection rates and detection of all large metastases, and allow for performance of unbiased size estimates.

Longitudinal MRI monitoring of brain damage in the neonatal ventral hippocampal lesion rat model of schizophrenia

Rat with excitotoxic neonatal ventral hippocampal lesions (NVHL rats) is considered as a heuristic neurodevelopmental model for studying schizophrenia. Extensive study of this model is limited by the lack of clear validity criteria of such lesions and because ascertaining of the lesions is realized postmortem with histological examination after completing experiments. Here, in a first experiment, by assessing the locomotor response to amphetamine in adult NVHL rats, we further specify that the lesions must be bilateral and confined to the ventral hippocampus to obtain the validated behavioral phenotype. We then show a longitudinal magnetic resonance imaging (MRI) protocol suitable for the detection of brain structural changes in NVHL rats. The T(2)-weighted images acquired in adult NVHL rats reveal the same structural changes as those appraised with histological protocol. Moreover, we demonstrate that the lesion status in adulthood can be accurately predicted from the T(2)-weighted images acquired in the juvenile period. As technical advantages, our MRI protocol makes possible to select animals according to lesion criteria as soon as in the juvenile period before long-lasting experiments and gives access in vivo to a quantitative parameter indicative of the lesion extent. Finally, we show that the lesion size increases only slightly between juvenile and adult periods. These latter results are discussed in the context of the specific postpubertal emergence of the behavioral deficits in NVHL rats.

Histological criteria for grading of atypia in melanocytic conjunctival lesions

To develop a standardised protocol for grading atypia in melanocytic conjunctival lesions (MCL), mainly primary acquired melanosis (PAM) and inflamed juvenile conjunctival naevi (IJCN) and to establish prognostic parameters for progression to malignant melanoma (MM). A retrospective non-randomised study of 304 patients with MCL was conducted. Histological slides of MCL diagnosed as conjunctival naevus (CN; 222 cases), PAM (42 cases), and IJCN (40 cases) were reviewed. A scoring method of atypia was developed according to the following histological parameters: nest cohesion, melanocytic hyperplasia, nuclear features, and pagetoid spread. Lesions were scored according to the number of criteria as mild (1-2), moderate (3-4) and severe (5-8). Nineteen PAM lesions showed mild (n = 8), moderate (n = 4), and severe (n = 7) atypia. The remaining PAM lesions were without atypia. IJCN cases showed architectural disorder only. Neither architectural disorder nor cytological atypia were found in the CN. There were no recurrences in the IJCN and PAM lesions without and with mild atypia, while two PAM with moderate and two with severe atypia recurred. Three cases (two of which had a history of recurrent PAM with severe atypia) progressed to MM. The criteria for evaluation of atypia were found to be significantly associated with the clinical outcome (p < 0.0001). The standardised histological scoring protocol of MCL is reliable, and may reduce the risk of confusing a benign MCL, such as IJCN, with one that is potentially preneoplastic. It may also prove useful in predicting which PAM lesions will progress to MM.

Developmental regulation of histone H3 methylation at lysine 4 in the porcine ovary

Follicular growth and oogenesis involve highly dynamic changes in morphogenesis, chromatin structure, and gene transcription. The tight coordination of these events leads to ovulation of a mature oocyte and formation of the luteal tissue necessary to regulate embryo implantation and development. This entire process is regulated by numerous endocrine and in situ mechanisms. The role of epigenetic mechanisms in folliculogenesis, such as the biochemical modification of the DNA packaging proteins, the histones, is not well understood. Our objective was to determine the cellular and follicular stage-specific patterns of histone H3 methylation at lysine 4 (K4) in porcine preovulatory follicles and during luteinization in pig ovaries. Ovary tissues were collected from slaughtered prepubertal and cyclic gilts at various stages of the estrous cycle, pregnancy, and from ovaries recovered from gonatropin-treated gilts at 0, 24, and 38 h post human chorionic gonadotropin (hCG) injection. Samples were fixed in 4% paraformaldehyde and processed for embedding in paraffin and sectioned using standard histological protocols. Immunofluorescent staining was performed on 3 microm thick sections. The immunostaining pattern of mono-, di-, and tri-methylated histone H3-K4 and lysine-specific demethylase 1 (LSD1, also known as KDM1 or AOF1) was assessed. Interestingly, H3-K4 mono-, di-, and tri-methylation in follicles of prepubertal gilts was specifically distributed and developmentally regulated. While granulosa cells of primary, secondary, and early antral follicles were negative for H3-K4 methylation those from large antral follicles showed a striking upregulation in the cells located in the proximity to the oocyte. Specifically, the cumulus oophorus displayed intense staining for H3-K4 methylation and signals were strongest in the granulosa cells in the inner two cell layers of the follicular wall. Although all oocytes from primary to large antral stage follicles were positive for H3-K4 mono-, di-, and tri-methylation, the patterns of distribution were altered through oocyte follicle development. H3-K4 methylation in granulosa cells was dramatically reduced as time to ovulation approached and was low to undetected at 38 h post hCG treatment. H3-K4 mono-, di-, and tri-methylation in large luteal cells increased as differentiation evolved but remained low in small luteal cells. Strikingly, LSD1 (KDM1) expression was found to be restricted to the corpus luteum. In summary, this study provides new information on histone H3-K4 methylation patterns in the oocyte and follicle during folliculogenesis, which suggests that these epigenetic markers serve an essential regulatory role during folliculogenesis.

Sentinel Lymph Nodes

Sentinel Lymph Nodes

Matrix Solution Fixation: Histology-Compatible Tissue Preparation for MALDI Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) acquires a grid of spatially resolved mass spectra and provides a molecular landscape of a tissue. This can have a myriad of uses: from basic tissue characterization to a comprehensive pathological diagnosis. We have developed a fast, inexpensive, histology-compatible tissue preparation method for matrix-assisted laser desorption/ionization (MALDI)-MSI, which overcomes current sample preparation-imposed limitations in image resolution. Tissue sections are prepared via simultaneous fixation and matrix deposition. This is accomplished by incorporating the MALDI matrix into solvents that preserve tissue integrity when applied according to standard histology procedures. This concept was expanded to include multiple histology protocols, thereby enabling analysis to be tailored to a variety of biomolecules and tissue types.