Histological Protocol Research Articles

Long‐Period Biorhythm Variation in Enamel Microstructure of Pre‐Columbian South Americans

It has been argued that the periodicity (in days) between dental enamel growth lines known as striae of Retzius reflects an autonomic biorhythm regulating metabolism and life history. Therefore, quantifying variation in Retzius periodicity (RP) has been recently used as a tool to provide life history and metabolic information among humans, primates, and other mammals. For example, body size is a major driving factor in life history evolution, and correlates strongly with RP in anthropoid primates but not lemurs.Varying datasets disagree as to whether this particular relationship among anthropoid species extends within species, particularly within modern humans. Moreover, little is known regarding the impact that geography has had on life history evolution among modern human populations. Altitude is one unexamined factor that may have influenced this evolution, as high‐altitude human populations exhibit multiple adaptations to their relatively hypoxic environment, and low oxygen availability has obvious consequences for overall metabolic output. Therefore, this study uses RP variation to compare a coastal (low altitude) and Andean (high altitude) sample of Pre‐Columbian South Americans. We have two major objectives: 1) determine if RP is correlated with body mass within this previously unsampled group, and more importantly; 2) determine if RP differs between coastal and Andean populations, a possible consequence of life history adaptations to altitude.To answer our questions, we gathered teeth from 10 individuals, 5 each from low and high altitude archaeological sites of Peru, all part of the anthropology collection at the American Museum of Natural History. Teeth were cleaned, embedded in acrylic resin, thin‐sectioned, and polished according to standard dental histology protocols. They were imaged in circularly polarized light using a Zeiss Axioskop II microscope. Photomicrographs of dental enamel were used to count the number of 24‐hour growth lines (cross‐striations) between striae of Retzius, indicating the number of days taken to form each successive stria (RP). An average of cross‐striation breadths was also divided into an average of striae of Retzius breadths in the same region of each tooth to verify the counted value. For each individual, orbital height and foramen magnum length were used as proxy variables to assess body size. Individuals were sexed based on standard cranial characteristics.Regressions of RP against body size proxies demonstrate a statistically significant negative correlation in the total combined sample. There is no significant difference in RP variation between the sexes, nor in body size between low and high altitude populations. However, the higher altitude population has significantly longer RP values. In conclusion, our analysis suggests that as predicted, an intraspecific relationship exists for RP and body size among our sample, and that altitude elicits as yet unidentified life history/metabolic differences expressed in RP, warranting further study. The high vs. low altitude pattern appears to be independent of the relationship we find between body size and RP in studies of other human populations.Support or Funding InformationFGCU Professional Development GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Polyethylene glycol fusion associated with antioxidants: A new promise in the treatment of traumatic facial paralysis.

Recent studies in invertebrates have taught us that early cell membrane regeneration is determinant for axonal recovery and survival after trauma. Many authors obtained extraordinary results in neural regeneration using polyethylene glycol fusion protocols, which also involved microsutures and antioxidants. Sixty rats were evaluated with functional and histological protocol after facial nerve neurotmesis. Groups A and B had their stumps coapted with microsuture after 24 hours of neurotmesis and groups C and D after 72 hours. In addition to the microstructure, groups B and D used the polyethylene glycol-fusion protocol for the modulation of the Ca+2 . At the sixth week, the latency of group D and duration of group B was lower than groups A and C (P = .011). The axonal diameter of the groups that used polyethylene glycol-fusion was higher than those who did not use polyethylene glycol-fusion (P ≤ .001). Although not providing a functional improvement, polyethylene glycol-fusion slowed down demyelination.

Histologic Analysis of Retrieved Synthetic Ligaments Implanted in Dogs for Treatment of Cranial Cruciate Ligament Disease

Objective: To histologically analyze retrieved synthetic liagments implanted in canine stifles for the treatment of cranial cruciate ligament disease.Animals: 6 client-owned dogsProcedures: Synthetic ligaments (SL) were retrieved from 6 dogs. Five had experienced postoperative complications which necessitated their removal and 1 dog died of unrelated causes. The formalin fixed SLs were stained with hematoxylin and eosin, Masson’s trichrome, Alcian blue, Vimentin and Reticulin stains using standard histologic staining protocols and examined histologically via normal light microscopy. The histologic evaluation involved both a qualitative description and semiquantitative assessment of each dog’s cellular response to the implanted SL. Additionally, a semiquantitative scoring system was used for describing the distribution and amount of fibroblasts around and within the central (inner 1/2) and peripheral (outer 1/2) zones and individual core fibers of the synthetic ligaments.Results: Sheath and core segments had amounts of cellular infiltration which ranged from minimal to moderate which consisted primarily of fibroblasts with rare multinucleate giant cells present and no evidence of lymphoid or inflammatory cellular infiltrates. Peripheral zone fibroblast ingrowth ranged from minimal to moderate and infiltration consistently decreased into the central (inner ½) zones. There was a minimal amount of fibroblast infiltration and encircling of individual core fibers. Infiltrating fibroblasts deposited collagen matrix and mild to moderate amounts of reticulin fibers within the hypercellular regions of the sheath with lesser amounts in the core segments.Conclusions and Clinical Relevance: The structure and composition of this synthetic ligament supported variable amounts of fibroblast ingrowth and activity while inducing a minimal amount of inflammatory cell infiltration.

Internal anal sphincter nerves - a macroanatomical and microscopic description of the extrinsic autonomic nerve supply of the internal anal sphincter.

The internal anal sphincter (IAS) contributes substantially to anorectal functions. While its autonomic nerve supply has been studied at the microscopic level, little information is available concerning the macroscopic topography of extrinsic nerve fibres. This study was designed to identify neural connections between the pelvic plexus and the IAS, provide a detailed topographical description, and give histological proof of autonomic nerve tissue. Macroscopic dissection of pelvic autonomic nerves was performed under magnification in seven (five male, two female) hemipelvises obtained from body donors (67-92years). Candidate structures were investigated by histological and immunohistochemical staining protocols to visualize nerve tissue. Nerve fibres could be traced from the anteroinferior edge of the pelvic plexus to the anorectal junction running along the neurovascular bundle anterolaterally to the rectum and posterolaterally to the prostate/vagina. Nerve fibres penetrated the longitudinal rectal muscle layer just above the fusion with the levator ani muscle (conjoint longitudinal muscle) and entered the intersphincteric space to reach the IAS. Histological and immunohistochemical findings confirmed the presence of nerve tissue. Autonomic nerve fibres supplying the IAS emerge from the pelvic plexus and are distinct to nerves entering the rectum via the lateral pedicles. Thus, they should be classified as IAS nerves. The identification and precise topographical location described provides a basis for nerve-sparing rectal resection procedures and helps to prevent postoperative functional anorectal disorders.

Histological Preparation Technique of Blood Derivative Injectable Platelet-Rich Fibrin (I-Prf) for Microscopic Analyzes

Objective: The objective of the present study was to describe the histological preparation technique of blood derivative injectable platelet-rich fibrin (i-PRF) for microscopic analyzes. Materials and methods: Blood samples were collected from 15 volunteers to prepare i-PRF samples. The difference between i-PRF and oral mucosa tissue experimental protocol for histological preparation was elaborated in Step 1 - fixation, with 01 hour for i-PRF fixation and 24 hours for oral mucosa tissue. The other histological preparation protocol steps used for the processing and staining of the specimens were similar to that used for oral mucosa tissue. Tissue and i-PRF samples were analyzed by immunofluorescence, immunohistochemistry and microscopic histology. Results: In comparing i-PRF and oral mucosal tissue, it was observed that both had similarities in morphology and coloration of the visualized structures. The technique demonstrates immunostaining regulation of TGF-β from i- PRF. VEGF was fully detectable using the immunofluorescence technique applied to i-PRF. The method showed a fundamental advantage in reducing the fixation time, since the established fixation time in oral mucosa tissue is at least 24 hours. For the i-PRF, it was observed that the fixation was 01 hour, so the reduced fixation time shows an advantage in reducing the total working time without compromising the quality of the analyzed samples. Conclusions: The method showed a distinct advantage in reducing the fixation time of i-PRF. These initial analyses revealed that the slow polymerization during i-PRF preparation generates a blood derivative with a special fibrin network rich in platelets and leukocytes, and the presence of immunostaining for TGF-β and immunofluorescence of VEGF.

Utility of WATS3D for Barrettʼs Esophagus in Community Practice

Introduction: Early detection is vital in determining a patient's risk for development of esophageal carcinoma. WATS3D (CDx Diagnostics, Suffern, NY) testing has been shown to increase the detection of Barrett's esophagus (BE) and low or high grade dysplasia by up to 40% when compared to the standard histological protocol. Methods: To determine the utility of WATS3D testing our community referral practice performed a retrospective review of patients over one year. The histological specimen result and the WATS3D result were recorded and compared. Patients previously diagnosed with BE as well as new patients were reviewed. We recorded BE, non- BE, dysplasia and indefinite dysplasia. The incremental increased detection rates for BE and dysplasia were noted for each patient. Results: One hundred and five patients underwent esophagogastroduodenoscopy with both WATS3D sampling and histologic testing. Eighty seven patients, or 83%, had surveillance exams for a previous BE diagnosis. Eighteen or 12.5% of the patients were new suspected BE. In the surveillance group, the original BE diagnosis was based on interpretation by pathologists with varied backgrounds and institutions. Fifty nine patients, or 68%, were confirmed to have BE by our testing. Eight of the 59 patients, or 13.6%, had BE detection by WATS3D only. Twenty eight patients, or 32%, of the surveillance group were negative for BE by both histology and WATS3D sampling. One surveillance patient had dysplasia findings noted on both histology and WATS3D sampling. One surveillance patient had indefinite dysplasia findings by WATS3D sampling only. In the newly suspected BE group 8 patients had a new diagnosis of BE made. Six diagnoses were made by histology and WATS3D sampling, 1 histology positive only and 1 WATS3. WATS3 sampling gave a 12.5% increase in detection. No dysplasia was noted on any newly diagnosed patients. No cancers were identified. Conclusion: WATS3D is a new technology that has the potential to increase diagnosis of BE with or without dysplasia. Utility in various populations is still being established. We report a 12.5% to 13.6% increase in BE detection in our community referral practice using WATS3D. The patient cost benefit ratio may vary in different patient populations and should be investigated further.

Mammary gland morphology in the northern Amazon red squirrel (Sciurus igniventris)

Sciurus igniventris is a squirrel with broad distribution in South America. We examined one female adult specimen which was donated after death by trampling. The aim of this work was describe the morphological characteristics of mammary gland of one specimen. The body was fixed using 10% aqueous formaldehyde. Tissues were processed and slides prepared following standard histological protocols. Morphological analysis of the mammary glands revealed similarities to those described in the literature for domestic and wild mammals. As special features in kind, macroscopically identified a pair of thoracic glands, followed by three pairs of abdominal glands. Microscopically the abundant presence of connective tissue dividing the alveolar-tubular glands. Similar to that described for domestic and wild mammals.

Local application of osteoprotegerin-chitosan gel in critical-sized defects in a rabbit model.

BackgroundOsteoprotegerin (OPG) is used for the systemic treatment of bone diseases, although it has many side effects. The aim of this study was to investigate a newly formulated OPG-chitosan gel for local application to repair bone defects. Recent studies have reported that immunodetection of osteopontin (OPN) and osteocalcin (OC) can be used to characterise osteogenesis and new bone formation.MethodsThe osteogenic potential of the OPG-chitosan gel was evaluated in rabbits. Critical-sized defects were created in the calvarial bone, which were either left unfilled (control; group I), or filled with chitosan gel (group II) or OPG-chitosan gel (group III), with rabbits sacrificed at 6 and 12 weeks. Bone samples from the surgical area were decalcified and treated with routine histological and immunohistochemical protocols using OC, OPN, and cathepsin K (osteoclast marker) antibodies. The toxicity of the OPG-chitosan gel was evaluated by biochemical assays (liver and kidney function tests).ResultsThe mean bone growth in defects filled with the OPG-chitosan gel was significantly higher than those filled with the chitosan gel or the unfilled group (p < 0.05). At 6 and 12 weeks, the highest levels of OC and OPN markers were found in the OPG-chitosan gel group, followed by the chitosan gel group. The number of osteoclasts in the OPG-chitosan gel group was lower than the other groups. The results of the liver and kidney functional tests indicated no signs of harmful systemic effects of treatment. In conclusion, the OPG-chitosan gel has many characteristics that make it suitable for bone repair and regeneration, highlighting its potential benefits for tissue engineering applications.

MP56-18 THE MOUSE CORPUS CAVERNOSUM GLANDUS IS BIOMECHANICALLY ANALOGOUS TO THE HUMAN CORPUS CAVERNOSUM

You have accessJournal of UrologySexual Function/Dysfunction: Peyronie’s Disease II1 Apr 2017MP56-18 THE MOUSE CORPUS CAVERNOSUM GLANDUS IS BIOMECHANICALLY ANALOGOUS TO THE HUMAN CORPUS CAVERNOSUM Matthew Hennefarth, Ling Chen, Ryan Hsi, Misun Kang, Amanda Reed-Maldonado, Guiting Lin, Marshall Stoller, Tom Lue, and Sunita Ho Matthew HennefarthMatthew Hennefarth More articles by this author , Ling ChenLing Chen More articles by this author , Ryan HsiRyan Hsi More articles by this author , Misun KangMisun Kang More articles by this author , Amanda Reed-MaldonadoAmanda Reed-Maldonado More articles by this author , Guiting LinGuiting Lin More articles by this author , Marshall StollerMarshall Stoller More articles by this author , Tom LueTom Lue More articles by this author , and Sunita HoSunita Ho More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1771AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Calcified Peyronie's plaque (CPP) is postulated to be of bone phenotype within a human penis. Rodents are used as models to investigate spatiotemporal mechanistic processes leading to CPP in humans; however, there are specific biomechanical differences between species. The comparable regions to program insults within rodents to induce the pathologically mineralized tissue similar to CPP are not well correlated. The objective of this study is to illustrate that the mouse corpus cavernosum glandus (CCG), instead of the mouse corpus cavernosum penis (CPP), is more analogous to the human corpus cavernosum (HCC). METHODS Penile tissues from 12 Scx-GFP mice with ages 4 week, 5 week, 6 week, and 1.2 years, were dissected and processed following routine histology protocols. 6μm thick sections were stained with H&E, Massons' trichrome (TRI), Verhoeff's elastin (ELA), and DAPI. All sections were imaged using bright field or fluorescence microscopy techniques. Cell orientations along collagen fibers with age were analyzed using Image J Fiji. RESULTS Figure: With age, CCG illustrated well-oriented struts at an angle of 32° to the baculum. Additionally, scleraxis and elastin expressions, and smooth muscle content increased within the struts, specifically in the 6 week-old mice. The expression of scleraxis indicated that the CCG contains mechanoactive collagenous and elastin-ridden struts that support biomechanical function of the baculum. Within the CCG of the mouse, there was more sinusoidal space in the younger age groups (4-6 weeks), and this space decreased with age, and elastin expression increased significantly (see 1 year). Compared to the CCG (A,C,E,G), the CCP (B,D,F,H) illustrated significantly lower expressions of scleraxis, elastin, and smooth muscle. CONCLUSIONS The collagen-producing fibroblasts within the CCG and CCP express scleraxis, indicative of local mechanoactive areas. Based upon orientation of cells expressing scleraxis, the biomechanical function of the CCP is to push the glans of the penis like a piston, while the CCG stiffens the penis during erection and pulls the glans of the penis back into the prepuce post-erection. The CCG is similar to the HCC with regard to compositional ratio of collagen to muscle, elastin localization, and biomechanical function and as such is a good model to study human penile physiology. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e761-e762 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Matthew Hennefarth More articles by this author Ling Chen More articles by this author Ryan Hsi More articles by this author Misun Kang More articles by this author Amanda Reed-Maldonado More articles by this author Guiting Lin More articles by this author Marshall Stoller More articles by this author Tom Lue More articles by this author Sunita Ho More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Sudden death in racehorses: postmortem examination protocol.

In racehorses, sudden death (SD) associated with exercise poses a serious risk to jockeys and adversely affects racehorse welfare and the public perception of horse racing. In a majority of cases of exercise-associated sudden death (EASD), there are no gross lesions to explain the cause of death, and an examination of the cardiovascular system and a toxicologic screen are warranted. Cases of EASD without gross lesions are often presumed to be sudden cardiac deaths (SCD). We describe an equine SD autopsy protocol, with emphasis on histologic examination of the heart ("cardiac histology protocol") and a description of the toxicologic screen performed in racehorses in California. By consistently utilizing this standardized autopsy and cardiac histology protocol, the results and conclusions from postmortem examinations will be easier to compare within and across institutions over time. The generation of consistent, reliable, and comparable multi-institutional data is essential to improving the understanding of the cause(s) and pathogenesis of equine SD, including EASD and SCD.

A novel morphological approach to gonads in disorders of sex development

AbstractDisorders of sex development are defined as congenital conditions with discordance between the phenotype, the genotype, the karyotype, and the hormonal profile. The disorders of sex development consensus classification established in 2005 are mainly based on chromosomal and biological data. However, histological anomalies are not considered. The aims of this study were to define the specific pathological features of gonads in various groups of disorders of sex development in order to clarify the nosology of histological findings and to evaluate the tumor risk in case of a conservative approach. One hundred and seventy-five samples from 86 patients with disorders of sex development were analyzed following a strict histological reading protocol. The term ‘gonadal dysgenesis' for the histological analysis was found confusing and therefore excluded. The concept of ‘dysplasia' was subsequently introduced in order to describe the architectural disorganization of the gonad (various degrees of irregular seminiferous tubules, thin albuginea, fibrous interstitium). Five histological types were identified: normal gonad, hypoplastic testis, dysplastic testis, streak gonad, and ovotestis. The analysis showed an association between undifferentiated gonadal tissue, a potential precursor of gonadoblastoma, and dysplasia. Dysplasia and undifferentiated gonadal tissue were only encountered in cases of genetic or chromosomal abnormality (‘dysgenesis' groups in the disorders of sex development consensus classification). ‘Dysgenetic testes', related to an embryonic malformation of the gonad, have variable histological presentations, from normal to streak. Conversely, gonads associated with hormonal deficiencies always display a normal architecture. A loss of expression of AMH and α-inhibin was identified in dysplastic areas. Foci of abnormal expression of the CD117 and OCT4 immature germ cells markers in dysplasia and undifferentiated gonadal tissue were associated with an increased risk of neoplasia. This morphological analysis aims at clarifying the histological classification and gives an indication of tumor risk of gonads in disorders of sex development.

Three-dimensional mapping of the arteriovenous loop model using two-dimensional histological methods.

The aim of this study was to create an analytical tool for the three-dimensional distribution of immunohistochemically stained cells in the arteriovenous (AV) loop model of the femoral vessels of rats that fuses two-dimensional histological slides into stacks. A total of 22 AV loops were implanted into male syngeneic Lewis rats by creating an arteriovenous fistula between the femoral artery and vein by interposing a femoral vein graft of the contralateral extremity. This fistula was embedded into an isolation chamber filled with a fibrin matrix. Specimens were explanted after 7 to 14 days, and the AV loop was processed using standard histological protocols. Immunohistochemical staining for HIF-1α and a counter staining with hematoxylin was performed. Various layouts with different cutting planes, regions of interest, and post-processing algorithms were evaluated. Results and observations: The proximal-to-distal cutting perpendicular to the vascular axis proved to be the best layout for mapping the three-dimensional constructs containing the AV loop. A semi-automatic algorithm for the differentiation of immunohistochemical positive and negative cells was developed. The newly established methods from this study constitute an excellent tool for the general evaluation of the AV loop model - particularly with regard to the three-dimensional distribution of immunohistochemical positive and negative cells.

Use of Polymerase Chain Reaction for Bivalve Pathogen Surveillance in the Yellow Clam Mesodesma mactroides.

The yellow clam Mesodesma mactroides is a valuable shellfish occurring from the southeastern coast of Brazil to the northern coast of Argentina. Populations of yellow clams are disappearing from their entire range, and the cause is still unknown. The objective of this paper was to search for World Organisation for Animal Health (OIE)-listed pathogens and their relatives in the genera Marteilia, Bonamia, and Perkinsus as well as Mikrocytos mackini and the virus OsHV-1 μ var the yellow clam population in southern Brazil using molecular techniques and classic histology protocols. A total of 180 clams were manually collected in the intertidal region at six sampling points covering the entire coast of Rio Grande do Sul State (length, 622 km) in 2013. Tissue samples were tested by OIE-recommended single-step conventional polymerase chain reaction assays. The screening showed no evidence of the specific sequences of the protistan parasites and viral pathogens at any site within the six zones under study. We recommend continuous monitoring of the mollusks in the region. Received July 3, 2015; accepted February 3, 2016.

Acute Cadmium Toxicity Induced Impairments in the Liver and Kidney of Freshwater Catfish, Heteropneustes fossilis (Bloch)

Background/objective: The heavy metal, Cadmium enters aquatic environment through natural / anthropogenic sources and exerts deleterious effects in fish. Hence, the present study is aimed at investigating the acute Cd toxicity induced histological alterations in the liver and kidney of catfish, Heteropneustes fossilis. Methods: The bioassay was performed in a static renewal regime with H. fossilis exposing to varying acute toxicity concentrations viz., 18.45, 36.9, 73.7, 147.4 and 294.8 mg/l for 96 h. The LC50 was determined to be 44.13 mg/l. Since 100 % mortality occurred in the highest concentrations after 96 h, liver and kidney tissues were collected from fish exposed to the first three lower concentrations. Standard histology protocol was followed to study the histological alterations. Findings: The histological alterations like increased kupffer cell and pycnotic nucleus, ruptured hepatic tissue and nucleus, cellular necrosis and focal necrosis were observed in the liver of fish exposed to Cd. Similarly, vacuolation, increased periglomerular and peritubular space, shrunken glomerulus, melanomacrophages and loss of cytoplasm were observed in the kidney of fish treated with Cd. The intensity of the histological alterations in both liver and kidney was found to be concentration and duration dependent. Hepatic tissues were found to be ruptured in the liver of fish exposed to all the three concentrations viz., 18.45, 36.9 and 73.7 mg Cd/l. The appearance of melanomacrophage aggregates is a generalized non-specific marker, indicating environmental stress. In the present study, melanomacrophages were observed in kidney of fish exposed to 36.9 and 73.7 mg Cd/l. Our present observations on histological alterations in the liver and kidney of H. fossilis were in conformity to the histological observations made in the liver and kidney of various fish species exposed to different toxicants. Applications: The histological alterations in liver and kidney H. fossilis induced by Cd suggest that Cd can be a potential toxicant and hence these alterations can be used as biomarkers to monitor aquatic pollution. Keywords: Acute Toxicity, Cadmium, Histology, Heteropneustes fossilis, Kidney, Liver

Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells.

The extraocular muscles (EOMs) comprise a group of highly specialized skeletal muscles controlling eye movements. Although a number of unique features of EOMs including their sparing in Duchenne muscular dystrophy have drawn a continuous interest, knowledge about these hard to reach muscles is still limited. The goal of this chapter is to provide detailed methods for the isolation and histological analysis of mouse EOMs. We first introduce in brief the basic anatomy and established nomenclature of the extraocular primary and accessory muscles. We then provide a detailed description with step-by-step images of our procedure for isolating (and subsequently cryosectioning) EOMs while preserving the integrity of their original structural organization. Next, we present several useful histological protocols frequently used by us, including: (1) a method for highlighting the general organization of periocular tissue, using the MyoD(Cre) × R26(mTmG) reporter mouse that elegantly distinguishes muscle (MyoD(Cre)-driven GFP(+)) from the non-myogenic constituents (Tomato(+)); (2) analysis by H&E staining, allowing for example, detection of the pathological features of the dystrophin-null phenotype in affected limb and diaphragm muscles that are absent in EOMs; (3) detection of the myogenic progenitors (i.e., satellite cells) in their native position underneath the myofiber basal lamina using Pax7/laminin double immunostaining. The EOM tissue harvesting procedure described here can also be adapted for isolating and studying satellite cells and other cell types. Overall, the methods described in this chapter should provide investigators the necessary tools for entering the EOM research field and contribute to a better understanding of this highly specialized muscle group and its complex micro-anatomy.

Accuracy and Adequacy of Computed Tomography–Guided Lung Biopsies: Experience From a Community Hospital

Small tissue biopsies obtained through minimally invasive methods have become the primary diagnostic tools for the pathologic characterization and testing of lung masses. In view of recent advances in targeted therapy for non-small cell lung carcinoma, and lung adenocarcinoma in particular, pathologists are now expected to thoroughly characterize lung lesions microscopically while making certain that enough tissue remains for potential molecular analysis if indicated. To report our experience with computed tomography (CT)-guided lung needle biopsies with particular concentration on diagnostic yield, diagnostic accuracy, and adequacy of tissue for molecular testing if indicated. A retrospective observational study analyzed 224 biopsies in 222 patients undergoing CT-guided lung needle biopsies. Accuracy of diagnosis and adequacy of tissue for molecular testing, if applicable, was evaluated. A standardized protocol for specimen evaluation, triage, and processing was used. This protocol included intraprocedural real-time microscopic specimen evaluation and triage by a pathologist and use of a histologic protocol specifically designed to conserve tissue for ancillary testing. The initial biopsy was considered successful if the specimen was malignant, had specific benign features, or had nonspecific benign features with follow-up supporting benign lesion. Initial biopsy failure cases were those with inadequate tissue or a nonspecific result with highly suspicious imaging or clinical findings. Of the 224 biopsies, 8 cases with benign but nonspecific findings lacked follow-up and were excluded from the study. The biopsy was diagnostically successful in 189 of 216 (88%) cases. Of these 189 cases, 154 (81%) were malignant, and 35 (19%) were benign. There were 28 diagnostic failures. Subsequent tissue sampling of 13 of 28 diagnostic failures found 9 (69%) to be malignant. Molecular studies were requested on 25 cases: 24 had sufficient material for some of the requested tests, and 20 had enough tissue for all requested testing. A standardized protocol and team approach for CT-guided lung needle biopsy optimizes the ability to achieve a high accurate diagnostic yield with adequate tissue for molecular testing.

Identifying Colonic Dysplasia and Cancer From Rectal Nuclear Nano-morphology Markers in Ulcerative Colitis Patients

Introduction: Current guidelines recommend that ulcerative colitis (UC) pts undergo annual surveillance colonoscopy with random colonic biopsies for detection of colorectal dysplasia and cancer. This is a suboptimal invasive technique for detecting all colorectal dysplasia. Less invasive surveillance measures that would stratify pts based on risk of developing dysplasia/cancer are needed. We hypothesize, based on the premise of cancer field effect, that markers from rectal biopsies can predict the presence of dysplastic/neoplastic lesions in the colon. This pilot study aims to evaluate whether Spatial-domain Low-coherence Quantitative Phase Microscopy (SL-QPM) derived nuclear nano-morphology markers from flat normal appearing rectal mucosa can detect remote colonic dysplasia/neoplasia in UC pts. Methods: We prospectively recruited UC pts undergoing surveillance colonoscopy with random colon biopsies as per the American Gastroenterological Association surveillance guidelines. Two extra biopsies from flat rectal mucosa were obtained, fixed in formalin and processed per standard histology protocol. According to the final histologic diagnoses from all random biopsies, pts were categorized into a lowrisk group, if pathology showed no dysplasia/cancer, or a high-risk group, if there was any dysplasia, including adenomatous tissue or colon adenocarcinoma. For each pt, SL-QPM analysis was performed on nuclei from 40 to 60 rectal columnar epithelial cells.Figure 1Results: A total of 148 pts were included; 127 low risk and 21 high risk. The high risk group included pts with colorectal cancer (n=1), low-grade dysplasia (n=5), high-grade dysplasia (n=1), indefinite dysplasia (n=2) or sporadic tubular adenoma (n=12). A set of nuclear nano-morphology markers distinguished low risk from high risk UC pts with high statistical significance. Results were not confounded by presence of active inflammation anywhere in the colon (p-value>0.5). The performance characterized by area under receiver operating characteristics curve is 0.82 in distinguishing low from high risk UC pts (86% sensitivity at 61% specificity). Conclusion: Nuclear nano-morphology markers from non-dysplastic rectal tissue identify remote colonic dysplasia in UC pts. This technology is unaffected by the presence of inflammation and stratifies patients into low versus high risk for harboring dysplasia/cancer. This approach could enhance the efficiency of dysplasia surveillance in UC patients and an ongoing study is underway to validate these findings.Figure 2Figure 3

A clearer view of the insect brain-combining bleaching with standard whole-mount immunocytochemistry allows confocal imaging of pigment-covered brain areas for 3D reconstruction.

In the study of insect neuroanatomy, three-dimensional (3D) reconstructions of neurons and neuropils have become a standard technique. As images have to be obtained from whole-mount brain preparations, pigmentation on the brain surface poses a serious challenge to imaging. In insects, this is a major problematic in the first visual neuropil of the optic lobe, the lamina, which is obstructed by the pigment of the retina as well as by the pigmented fenestration layer. This has prevented inclusion of this major processing center of the insect visual system into most neuroanatomical brain atlases and hinders imaging of neurons within the lamina by confocal microscopy. It has recently been shown that hydrogen peroxide bleaching is compatible with immunohistochemical labeling in insect brains, and we therefore developed a simple technique for removal of pigments on the surface of insect brains by chemical bleaching. We show that our technique enables imaging of the pigment-obstructed regions of insect brains when combined with standard protocols for both anti-synapsin-labeled as well as neurobiotin-injected samples. This method can be combined with different fixation procedures, as well as different fluorophore excitation wavelengths without negative effects on staining quality. It can therefore serve as an effective addition to most standard histology protocols used in insect neuroanatomy.

Abstract 5254: Sentinel lymph node detection and in vivo/ex vivo assessment of melanin distribution by means of multispectral optoacoustic tomography (MSOT) in patients with malignant melanoma

Abstract Melanoma accounts for less than 5% of skin cancer cases, yet it causes more than 75% of skin cancer death, and its incidence is growing faster than any other cancer in the world. Because melanoma metastasizes early into regional/sentinel lymph nodes (SLN), SLN excision (SLNE) is probably the most important diagnostic procedure for melanoma patients, as histology provides the most relevant prognostic factor for the survival of melanoma patients. However, 50% of excised lymph nodes show no evidence of metastasis; and, current histological protocols involve sampling only a small portion of the SLN, resulting in a relatively high false negative rate. Furthermore, SLNE exposes the patients to complications such as swelling, edema and future risk of infection. Therefore, a clear need exists to improve the sensitivity and specificity of SLN analysis. Multispectral optoacoustic tomography (MSOT) ulitizes the molecular specificity of optical imaging, while capitalizing on the high temporal and spatial resolution of ultrasound imaging. This method allows sensitive detection of optical markers such as melanin, which would identify potentially metastatic lymph nodes, and indocyanine green (ICG), which can potentially label SLN. In this study, 148 lymph nodes were excised from 65 melanoma patients (stage I - IV), and the lymph nodes were examined by MSOT ex vivo to guide the pathologist to examination of melanin-containing regions of the lymph node, thereby increasing the detection rate in histological analysis. Compared to standard histology, MSOT demonstrated a superior 100% sensitivity/47% specificity. Further, 21 melanoma patients were scanned with a handheld MSOT device in vivo. MSOT was able to detect sentinel lymph nodes using ICG specific contrast, with the added ability of non-invasive assessment of the melanin status prior to excision. In vivo MSOT measurements using a 2D and a 3D detector were compared with ultrasound, SPECT/CT, planar fluorescence imaging and ex vivo histology, with a promising concordance between in vivo MSOT measurements and in vivo and ex vivo gold standard assessments. MSOT represents a viability modality to improve histological analysis of excised SLN in melanoma patients, and it offers the ability to stage lymph nodes noninvasively, potentially reducing the necessity to excise lymph nodes in some melanoma patients. Citation Format: Stefan Morscher, Ingo Stoffels, Neal C. Burton, Jing Claussen, Thomas Sardella, Iris Helfrich, Uwe Hillen, Julia Leyh, Dirk Schadendorf, Matthias Gunzer, Joachim Klode. Sentinel lymph node detection and in vivo/ex vivo assessment of melanin distribution by means of multispectral optoacoustic tomography (MSOT) in patients with malignant melanoma. [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 5254. doi:10.1158/1538-7445.AM2015-5254

Alterations in Hoffa's fat pad induced by an inflammatory response following idealized anterior cruciate ligament surgery.

To determine whether inflammation following anterior cruciate ligament (ACL) reconstruction leads to long-term pathological changes in the infrapatellar fat pad (IPFP or Hoffa's fat pad) which could compromise the integrity of the knee joint. Sixteen mature sheep underwent anatomic idealized ACL reconstruction surgery (ACL-R) and were sacrificed at 2 weeks (n = 9) and 20 weeks (n = 7) post-ACL-R. Five additional animals served as unoperated controls. A histological grading protocol was developed to quantify the changes in the IPFP post-injury. mRNA expression levels for key markers of inflammation, angiogenesis and tissue regeneration were assessed by qPCR. The IPFP exhibited altered cellularity and fibrosis at 2 and 20 weeks post-ACL-R. Immunohistochemistry detected macrophage-like cells in the IPFP which were increased at 20 weeks. Specific pro-inflammatory cytokines and IPFP specific adipokines exhibited changes indicating early inflammation mediated alterations. Elevations in CD105 mRNA levels at 2 weeks corroborated the increases in neovascularization observed in the IPFP following injury. Sustained long-term pathological changes stemming from inflammation are present in IPFP tissue after ACL-R surgery and may compromise the long-term integrity of the knee joint.