Tissue Preparations Research Articles

Safe and efficient dental cavity preparation by applying a gradient-in-power approach from Er,Cr:YSGG laser: an in-vitro study

In this in-vitro study, a gradient-in-power approach aims to ensure no temperature elevation beyond the necrosis limit (5.5 °C) during laser cavity preparation of dental hard tissues. The applied optimal Er,Cr:YSGG laser parameters were: 20 Hz pulse repetition rates (prr), average powers at a maximum of 5.5 W for enamel switched to a maximum of 3.5 W for dentine surface specimens. A fabricated fast-response all-optical fiber sensor was used to monitor temperature change simultaneously. A scanning electron microscope (SEM) and a Fourier transform infrared (FTIR) spectroscopy were used to assess the irradiated surfaces. Holes of 500 µm in depth were obtained with no morphological and chemical alterations.

The roles of serine hydrolases and serum albumin in alisol B 23-acetate hydrolysis in humans.

Introduction: Alisol B 23-acetate (AB23A), a major bioactive constituent in the Chinese herb Zexie (Rhizoma Alismatis), has been found with multiple pharmacological activities. AB23A can be readily hydrolyzed to alisol B in mammals, but the hydrolytic pathways of AB23A in humans and the key enzymes responsible for AB23A hydrolysis are still unrevealed. This study aims to reveal the metabolic organs and the crucial enzymes responsible for AB23A hydrolysis in human biological systems, as well as to decipher the impact of AB23A hydrolysis on its biological effects. Methods: The hydrolytic pathways of AB23A in human plasma and tissue preparations were carefully investigated by using Q-Exactive quadrupole-Orbitrap mass spectrometer and LC-UV, while the key enzymes responsible for AB23A hydrolysis were studied via performing a set of assays including reaction phenotyping assays, chemical inhibition assays, and enzyme kinetics analyses. Finally, the agonist effects of both AB23A and its hydrolytic metabolite(s) on FXR were tested at the cellular level. Results: AB23A could be readily hydrolyzed to form alisol B in human plasma, intestinal and hepatic preparations, while human butyrylcholinesterase (hBchE) and human carboxylesterases played key roles in AB23A hydrolysis in human plasma and tissue preparations, respectively. It was also found that human serum albumin (hSA) could catalyze AB23A hydrolysis, while multiple lysine residues of hSA were covalently modified by AB23A, suggesting that hSA catalyzed AB23A hydrolysis via its pseudo-esterase activity. Biological tests revealed that both AB23A and alisol B exhibited similar FXR agonist effects, indicating AB23A hydrolysis did not affect its FXR agonist effect. Discussion: This study deciphers the hydrolytic pathways of AB23A in human biological systems, which is very helpful for deep understanding of the metabolic rates of AB23A in humans, and useful for developing novel prodrugs of alisol B with desirable pharmacokinetic behaviors.

Abstract 4698: Reproducibility in spatial biology: reducing variables to improve the reliability of insight generation

Abstract There is an ongoing crisis in reproducibility in scientific studies, and studies in spatial biology are no exception. Cell DIVETM Multiplex imaging solution (Leica Microsystems) provides reliable workflow solutions to minimize variability from study to study. Cell DIVE allows probing and imaging of dozens of biomarkers on a whole tissue section with an iterative staining and dye inactivation workflow. At its core, Cell DIVE is designed to provide methods reproducibility, from tissue preparation, antigen retrieval, and sample imaging and slide storage. Cell DIVE is designed to work with directly conjugated primary antibodies, another source of variability. However, conjugated antibodies from Cell Signaling Technology (CST) are rigorously validated using stringent acceptance criteria to reduce variability. In addition, the use of recombinant antibodies, the consistent conjugate brightness and antibody degree-of-labeling reduce lot to lot variability, ensuring reliable conjugated antibodies for spatial biology studies. High resolution imaging results are obtained by consistent round to round imaging, consistent calibration and corrections, and reduction of human error using the BAB 200 liquid handling solution (Advanced Solutions Life Sciences). We present here, an iterative biomarker study using adjacent tissue sections probed with distinct lots of CST antibody panels, imaged in temporally separated batches using a Cell DIVE imager fitted with a BAB200 liquid handler. Following robust analysis (segmentation, phenotyping and statistical analysis), we report the reproducibility findings across parameters. Methods and results reproducibility in spatial biology is essential for reliable insight generation, giving confidence in the quality of future studies aimed at improving patient outcomes. Citation Format: Lisa Arvidson, Reginaldo Prioli, Samuel Jensen, James B. Hoying, Michael W. Golway, Michael J. Smith, Katie O. White, Richard A. Heil-Chapdelaine, Chi-Chou Huang, Tuan H. Phan, Hideki Sasaki, Melinda Angus-Hill. Reproducibility in spatial biology: reducing variables to improve the reliability of insight generation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4698.

Inhibition of Gluconeogenesis by Boldine in the Perfused Liver: Therapeutical Implication for Glycemic Control.

The alkaloid boldine occurs in the Chilean boldo tree (Peumus boldus). It acts as a free radical scavenger and controls glycemia in diabetic rats. Various mechanisms have been proposed for this effect, including inhibited glucose absorption, stimulated insulin secretion, and increased expression of genes involved in glycemic control. Direct effects on glucose synthesis and degradation were not yet measured. To fill this gap, the present study is aimed at ensuring several metabolic pathways linked to glucose metabolism (e.g., gluconeogenesis) in the isolated perfused rat liver. In order to address mechanistic issues, energy transduction in isolated mitochondria and activities of gluconeogenic key enzymes in tissue preparations were also measured. Boldine diminished mitochondrial ROS generation, with no effect on energy transduction in isolated mitochondria. It inhibited, however, at least three enzymes of the gluconeogenic pathway, namely, phosphoenolpyruvate carboxykinase, fructose-bisphosphatase-1, and glucose 6-phosphatase, starting at concentrations below 50 μM. Consistently, in the perfused liver, boldine decreased lactate-, alanine-, and fructose-driven gluconeogenesis with IC50 values of 71.9, 85.2, and 83.6 μM, respectively. Conversely, the compound also increased glycolysis from glycogen-derived glucosyl units. The hepatic ATP content was not affected by boldine. It is proposed that the direct inhibition of hepatic gluconeogenesis by boldine, combined with the increase of glycolysis, could be an important event behind the diminished hyperglycemia observed in boldine-treated diabetic rats.

Abstract 5357: Estrogen receptor gene expression prediction from H&E-stained whole slide images

Abstract Gene expression profiling (GEP) represents an important approach to inform breast cancer treatment. However, access to GEP involves challenges associated with cost, tissue transportation, and turn around time. In this work, we explore the prediction of estrogen receptor gene (ESR1) expression directly from images of hematoxylin and eosin (H&E) stained, formalin-fixed paraffin-embedded (FFPE) breast cancer tissue. Since H&E staining is a fast and inexpensive component of the standard tissue preparation in pathology, this approach is tissue preserving and requires no additional tissue processing. Our method uses a deep multiple instance learning approach to process cropped image patches from whole-slide images (WSI) into a numeric embedding vector summarizing the information in each patch. A gated attention mechanism then aggregates these embeddings into a single prediction for the WSI. We train and tune the model on a site-based split of The Cancer Genome Atlas (TCGA) BRCA dataset, and evaluate it on both a heldout split of TCGA (independent sites) and a separate dataset from a tertiary teaching hospital (TTH). All splits of TCGA have ESR1 value, immunohistochemistry (IHC) estrogen receptor (ER) status, and limited clinical outcome data. The TTH dataset has IHC-based ER status and clinical outcome, but not ESR1 expression. On the TCGA heldout test split, our model’s root mean square error (RMSE) for predicting normalized gene expression counts (TPM) was 2.90 [95% CI: 2.57, 3.23], and the Pearson correlation was 0.57 [95%CI; 0.46, 0.67]. For predicting IHC-based ER status on the same TCGA split, this weakly-supervised ESR1-predicting model had an area under the receiver-operator curve (AUROC) of 0.81 [0.74, 0.87]. This was comparable to a strongly-supervised method directly predicting ER status (AUROC: 0.85 [0.77, 0.92]). Lastly, when evaluated for association with patient outcomes (progression-free interval; PFI) using the independent TTH dataset, this ESR1-predicting model had a concordance index (c-index) of 0.59 [0.52, 0.65]. For comparison, the c-index for PFI using the IHC-based ER status for these cases was 0.61 [0.54, 0.66]. This work further demonstrates the potential to infer gene expression from H&E stained images in a manner that shows meaningful associations with clinical variables. Because obtaining H&E stained images is substantially easier and faster than genetic testing, the capability to derive molecular genetic information from these images may increase access to this type of information for patient risk stratification and provide research insights into molecular-morphological associations. Future work incorporating more comprehensive sets of genes remains a valuable next step. Citation Format: Anvita A. Srinivas, Ronnachai Jaroensri, Ellery Wulczyn, James H. Wren, Elaine E. Thompson, Niels Olson, Fabien Beckers, Melissa Miao, Yun Liu, Cameron Chen, David F. Steiner. Estrogen receptor gene expression prediction from H&E-stained whole slide images. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5357.

Expression of Osterix and SOX9 after Administration of Gourami Fish Scale Collagen in Wistar Rats

The current goal of periodontal treatments is to regenerate periodontal tissue by adding bone grafts. Various materials are being explored as bone substitutes, including type 1 collagen. Gourami fish scales are known to be an alternative source of type 1 collagen and have potential as a substitute material for bone grafts. The objective of this study was to determine whether there is an increase in the expression of Osterix (OSX) and SOX9 after the administration of gourami (Osphronemus goramy) fish scale collagen in extracted socket Wistar rats (Rattus norvegicus). The research was conducted on 32 male Wistar rats that were classified into four groups: control groups on day 7 (K7) and day 14 (K14); and treatment groups on day 7 (P7) and day 14 (P14). The expression of OSX and SOX9 was visualized by immunohistochemical staining of bone tissue preparations by anti-OSX and SOX9 monoclonal antibodies. The ANOVA results for OSX expression had a significant difference in OSX expression between that in the control and treatment groups on day 7 and day 14. The ANOVA results for SOX9 expression on day 7 had a significant difference in SOX9 expression between that in the control and treatment groups. ANOVA results for SOX9 expression on day 14 had no significant difference in the expression of SOX9 between the treatment and control groups. The conclusion of this study was administration of Gourami scale collagen can increase the expression of OSX and SOX9 in socket’s Wistar rats. Keywords: Bone grafts, Type 1 collagen, Gourami fish scales, Osterix, SOX9

The Lions Eye Bank of Western Australia: 35 Years of Eye Banking (1986–2021)

Purpose: The purpose of this study was to review the trends in preparation, storage, and usage of donor corneal tissue from 35 years of eye banking at the Lions Eye Bank of Western Australia (LEBWA). Methods: Donor and recipient records for the period July 1986 to July 2021 were evaluated with relation to donor demographics, cause of death, storage medium, corneal utilization, type of corneal graft, and indication for graft. Results: Six thousand two hundred nine eyes from 3328 donors were retrieved in the 35-year period reviewed. Sixty-six percent of eye donors were male, and 33% were female. The median age of donor was 61 years (2–98 years). The most frequent cause of donor death was a cardiac event, followed by intracerebral hemorrhage and malignancy. A total of 5219 corneal transplants were performed, with a corneal utilization rate of 84%. Storage of corneal tissue was primarily in Optisol-GS until 2010, when organ culture medium was introduced. Of 5219 recipients, 52% were male and 47% were female. The median recipient age was 63 years (1–98 years). Penetrating keratoplasty was the most common surgery performed over the 35-year period, followed by endothelial keratoplasty. The most common indications for grafts were ectasia/thinning, repeat corneal transplants, endothelial dystrophies, and postcataract surgery edema. Conclusions: We present trends in the acquisition and utilization of corneal tissue across 35 years at LEBWA. The most significant changes over this period include methods of corneal storage and advances in surgical techniques that has led to a greater usage of endothelial grafts.

Lipidomic Analysis of Mouse Brain to Evaluate the Efficacy and Preservation of Different Tissue Preparatory Techniques by IR-MALDESI-MSI

Numerous preparatory methods have been developed to preserve the cellular and structural integrity of various biological tissues for different -omics studies. Herein, two preparatory methods for mass spectrometry imaging (MSI) were evaluated, fresh-frozen and sucrose-embedded, paraformaldehyde (PFA) fixed, in terms of ion abundance, putative lipid identifications, and preservation of analyte spatial distributions. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI)-MSI was utilized to compare the preparatory methods of interest with and without the use of the conventional ice matrix. There were 2.5-fold and 1.6-fold more lipid species putatively identified in positive- and negative-ion modes, respectively, for sucrose-embedded, PFA-fixed tissues without an ice matrix relative to the current IR-MALDESI-MSI gold-standard, fresh-frozen tissue preparation with an exogenous ice matrix. Furthermore, sucrose-embedded tissues demonstrated improved spatial distribution of ions resulting from the cryo-protective property of sucrose and paraformaldehyde fixation. Evidence from these investigations supports sucrose-embedding without ice matrix as an alternative preparatory technique for IR-MALDESI-MSI.

Rational Construction of a Novel Bioluminescent Substrate for Sensing the Tumor-Associated Hydrolase Notum.

Notum, one of the key serine hydrolases in mammals, hydrolyzes the palmitoleoyl moieties of many important proteins and modulates multiple signaling pathways including Wnt/β-catenin signaling. Notum is tightly associated with multiple human diseases, but the reliable and practical tools for sensing Notum activities in complex biological systems are rarely reported. Herein, an efficient strategy was used to rationally construct a specific bioluminescent substrate for Notum. Following computer-aided molecular design and experimental verification, octanoyl luciferin (OL) was selected as the optimum substrate for human Notum, with excellent specificity, high detection sensitivity and high signal-to-noise ratio. Under physiological conditions, OL was readily hydrolyzed by Notum or Notum-containing biological specimens to release d-luciferin that could be easily detected by various fluorescence devices in the presence of luciferase. The applicability of OL for real-time sensing native Notum was examined in living cells, extracellular matrix, and tissue preparations. OL was also used for constructing a high-throughput assay for screening of Notum inhibitors, while a natural compound (bergapten) was newly identified as a potent Notum inhibitor. Collectively, this study devises a reliable and easy-to-use tool for sensing Notum activities in biological systems, which will strongly facilitate hNotum-associated fundamental studies, disease diagnosis, and drug discovery.

Emerging technology for intraoperative margin assessment and post-operative tissue diagnosis for breast-conserving surgery

IntroductionTissue-preserving surgery is utilized progressively in cancer therapy, where a clear surgical margin is critical to avoid cancer recurrence, specifically in breast cancer (BC) surgery. The Intraoperative pathologic approaches that rely on tissue segmenting and staining have been recognized as the ground truth for BC diagnosis. Nevertheless, these methods are constrained by its complication and timewasting for tissue preparation. ObjectiveWe present a non-invasive optical imaging system incorporating a hyperspectral (HS) camera to discriminate between cancerous and non-cancerous tissues in ex-vivo breast specimens, which could be an intraoperative diagnostic technique to aid surgeons during surgery and later a valuable tool to assist pathologists. MethodsWe have established a hyperspectral Imaging (HSI) system comprising a push-broom HS camera at wavelength 380∼1050 nm with source light 390∼980 nm. We have measured the investigated samples' diffuse reflectance (Rd), fixed on slides from 30 distinct patients incorporating mutually normal and ductal carcinoma tissue. The samples were divided into two groups, stained tissues during the surgery (control group) and unstained samples (test group), both captured with the HSI system in the visible and near-infrared (VIS-NIR) range. Then, to address the problem of the spectral nonuniformity of the illumination device and the influence of the dark current, the radiance data were normalized to yield the radiance of the specimen and neutralize the intensity effect to focus on the spectral reflectance shift for each tissue. The selection of the threshold window from the measured Rd is carried out by exploiting the statistical analysis by calculating each region's mean and standard deviation. Afterward, we selected the optimum spectral images from the HS data cube to apply a custom K-means algorithm and contour delineation to identify the regular districts from the BC regions. ResultsWe noticed that the measured spectral Rd for the malignant tissues of the investigated case studies versus the reference source light varies regarding the cancer stage, as sometimes the Rd is higher for the tumor or vice versa for the normal tissue. Later, from the analysis of the whole samples, we found that the most appropriate wavelength for the BC tissues was 447 nm, which was highly reflected versus the normal tissue. However, the most convenient one for the normal tissue was at 545 nm with high reflection versus the BC tissue. Finally, we implement a moving average filter for noise reduction and a custom K-means clustering algorithm on the selected two spectral images (447, 551 nm) to identify the various regions and effectively-identified spectral tissue variations with a sensitivity of 98.95%, and specificity of 98.44%. A pathologist later confirmed these outcomes as the ground truth for the tissue sample investigations. ConclusionsThe proposed system could help the surgeon and the pathologist identify the cancerous tissue margins from the non-cancerous tissue with a non-invasive, rapid, and minimum time method achieving high sensitivity up to 98.95%.

Development of a full-thickness acellular dermal graft from human skin: Case report of first patient rotator cuff patch augmentation repair

The processing and initial testing of a new human tissue preparation is described. Full-thickness Acellular Dermal Matrix (ftADM) is the extracellular matrix (ECM) obtained by decellularization of full-thickness human skin from cadaveric donors. The safety, stability and usability of the graft are discussed with respect to the results of the residual cellular content, maintenance of ECM components, and biomechanical properties. Quantitative and qualitative analysis of the ECM demonstrated the absence of cell debris, while the native structure of human dermis was maintained. Biomechanical testing showed stiffness values comparable to other commercial products used for tendon reinforcement, suggesting that our ftADM could be successfully used not only in soft tissue regeneration surgeries, but also in tendon reinforcement. First case of ftADM in rotator cuff augmentation is described. Technical management of the patch during surgery and clinical outcomes are discussed.

Fiber enhancement and 3D orientation analysis in label-free two-photon fluorescence microscopy

Fluorescence microscopy can be exploited for evaluating the brain’s fiber architecture with unsurpassed spatial resolution in combination with different tissue preparation and staining protocols. Differently from state-of-the-art polarimetry-based neuroimaging modalities, the quantification of fiber tract orientations from fluorescence microscopy volume images entails the application of specific image processing techniques, such as Fourier or structure tensor analysis. These, however, may lead to unreliable outcomes as they do not isolate myelinated fibers from the surrounding tissue. In this work, we describe a novel image processing pipeline that enables the computation of accurate 3D fiber orientation maps from both grey and white matter regions, exploiting the selective multiscale enhancement of tubular structures of varying diameters provided by a 3D implementation of the Frangi filter. The developed software tool can efficiently generate orientation distribution function maps at arbitrary spatial scales which may support the histological validation of modern diffusion-weighted magnetic resonance imaging tractography. Despite being tested here on two-photon scanning fluorescence microscopy images, acquired from tissue samples treated with a label-free technique enhancing the autofluorescence of myelinated fibers, the presented pipeline was developed to be employed on all types of 3D fluorescence images and fiber staining.

Virtual Fluorescence Translation for Biological Tissue by Conditional Generative Adversarial Network.

The online version contains supplementary material available at 10.1007/s43657-023-00094-1.

Optimal Preparation of Adipose Tissue in Frozen Sections for Mohs Micrographic Surgery.

Huerta, Tomas MD*; Bailey, Alison MD*; Bergin, Jarrett BA*; Cook, Joel MD* Author Information

Outcome of Surgical Fixation for Midfoot Charcot Neuroarthropathy - A Systematic Review.

Charcot arthropathy is a condition which is progressive, non-infectious, destructive and debilitating that commonly affect foot and ankle. This systematic review is to evaluate the occurrence of common outcomes associated with each intervention of Charcot neuroarthropathy in midfoot. A systematic review on literatures that were published from Jan 2010 to Jan 2020 were collected, reviewed and selected regarding the surgical treatment procedures of Charcot neuroarthropathy in midfoot. The initial search yielded 231 reports and after exclusion, nine out of the total studies were included in the outcome analysis for review. These were studies that included data concerning surgical reconstruction of Charcot arthropathy in the midfoot. It is suggested that soft tissue preparation and usage of combination of implants thus reduce the risk of infection as well as increase rigidity of construct, respectively. These factors will aid to improve outcome of midfoot Charcot arthropathy reconstruction.

108P Establishing a multi-modal tissue preparation and imaging workflow to study heterogeneity in neuroblastoma tumors

108P Establishing a multi-modal tissue preparation and imaging workflow to study heterogeneity in neuroblastoma tumors

Improvements and challenges of tissue preparation for spatial transcriptome analysis of skull base tumors

BackgroundSpatial transcriptome (ST) provides molecular profiles of tumor cells at the spatial level, which brings new progress to the research of tumors and the tumor microenvironment. This study summarizes the experiences and lessons learned in the spatial section preparation of two different pathological types of nose and skull base tumors at our institution, with the aim of offering guidelines to researchers to avoid wasting precious samples and provide a basis for the application of ST in clinical practice. MethodsFrozen tissue blocks from patients with squamous cell carcinoma and adenocarcinoma of the nose and skull base diagnosed at our institution were prepared. The effects of different procedures and pathological tissue types on slide quality were explored and evaluated using RNA integrity number (RIN) and HE scores as criteria. The effects of different RIN values on ST sequencing data were explored. ResultsA total of 43 samples were obtained from 26 patients, including 22 with squamous carcinomas and 21 with adenocarcinomas. Thirteen samples with satisfactory RNA quality control and good histological morphology were sequenced for ST. Sample isolation time <15 min and abandonment of snap-frozen isopentane significantly improved RNA quality (p = 0.004, p < 0.0001) and histomorphological integrity (p = 0.02, p = 0.02). Selection of a suitable tissue RNA extraction kit was critical for RNA quality (p < 0.0001). No difference between 6 ≤ RIN <7 and RIN >7 in ST sequencing results was found, indicating that RIN ≥6 can be used as a criterion for qualified RNA quality control. Therefore, fresh tissues washed as soon as possible with cold PBS and then dried using OCT for snap freezing are currently the best method for preparing spatial sections of nose and skull base tumor tissues of different pathological types. ConclusionThis study is the first to investigate the feasibility of applying ST to different pathological types of nose and skull base tumors and to demonstrate the widespread application of ST in tumors. Rational optimization of spatial slide preparation procedures and exploration of individualized pre-sequencing protocols are used as the first stage to ensure the quality of spatial sequencing and lay the foundation for subsequent spatial analysis.

Intraoperative confocal laser endomicroscopy: prospective in vivo feasibility study of a clinical-grade system for brain tumors.

The authors evaluated the feasibility of using the first clinical-grade confocal laser endomicroscopy (CLE) system using fluorescein sodium for intraoperative in vivo imaging of brain tumors. A CLE system cleared by the FDA was used in 30 prospectively enrolled patients with 31 brain tumors (13 gliomas, 5 meningiomas, 6 other primary tumors, 3 metastases, and 4 reactive brain tissue). A neuropathologist classified CLE images as interpretable or noninterpretable. Images were compared with corresponding frozen and permanent histology sections, with image correlation to biopsy location using neuronavigation. The specificities and sensitivities of CLE images and frozen sections were calculated using permanent histological sections as the standard for comparison. A recently developed surgical telepathology software platform was used in 11 cases to provide real-time intraoperative consultation with a neuropathologist. Overall, 10,713 CLE images from 335 regions of interest were acquired. The mean duration of the use of the CLE system was 7 minutes (range 3-18 minutes). Interpretable CLE images were obtained in all cases. The first interpretable image was acquired within a mean of 6 (SD 10) images and within the first 5 (SD 13) seconds of imaging; 4896 images (46%) were interpretable. Interpretable image acquisition was positively correlated with study progression, number of cases per surgeon, cumulative length of CLE time, and CLE time per case (p ≤ 0.01). The diagnostic accuracy, sensitivity, and specificity of CLE compared with frozen sections were 94%, 94%, and 100%, respectively, and the diagnostic accuracy, sensitivity, and specificity of CLE compared with permanent histological sections were 92%, 90%, and 94%, respectively. No difference was observed between lesion types for the time to first interpretable image (p = 0.35). Deeply located lesions were associated with a higher percentage of interpretable images than superficial lesions (p = 0.02). The study met the primary end points, confirming the safety and feasibility and acquisition of noninvasive digital biopsies in all cases. The study met the secondary end points for the duration of CLE use necessary to obtain interpretable images. A neuropathologist could interpret the CLE images in 29 (97%) of 30 cases. The clinical-grade CLE system allows in vivo, intraoperative, high-resolution cellular visualization of tissue microstructure and identification of lesional tissue patterns in real time, without the need for tissue preparation.

Diagnostic Challenges and Solutions in Systemic Amyloidosis

Amyloidosis refers to a clinically heterogeneous group of disorders characterized by the extracellular deposition of amyloid proteins in various tissues of the body. To date, 42 different amyloid proteins that originate from normal precursor proteins and are associated with distinct clinical forms of amyloidosis have been described. Identification of the amyloid type is essential in clinical practice, since prognosis and treatment regimens both vary according to the particular amyloid disease. However, typing of amyloid protein is often challenging, especially in the two most common forms of amyloidosis, i.e., the immunoglobulin light chain amyloidosis and transthyretin amyloidosis. Diagnostic methodology is based on tissue examinations as well as on noninvasive techniques including serological and imaging studies. Tissue examinations vary depending on the tissue preparation mode, i.e., whether it is fresh-frozen or fixed, and they can be carried out by ample methodologies including immunohistochemistry, immunofluorescence, immunoelectron microscopy, Western blotting, and proteomic analysis. In this review, we summarize current methodological approaches used for the diagnosis of amyloidosis and discusses their utility, advantages, and limitations. Special attention is paid to the simplicity of the procedures and their availability in clinical diagnostic laboratories. Finally, we describe new methods recently developed by our team to overcome limitations existing in the standard assays used in common practice.

Testicular histopathology in rats co-exposed to heat and psychological stressors

ObjectivesThe present study aimed to investigate the effect of co-exposure to heat and psychological stressors on testicular tissue as one of the major male reproductive organs in rats. MethodsForty adult male Wistar rats were divided into four groups of 10 animals each. The first group was exposed to heat stress (Temperature Humidity Index: 57.75 ± 3), the second group was exposed to three psychological stressors including strobe light ultrasonic sound, and tilting cage, and the third group was co-exposed to both heat and psychological stress daily. The order of exposure to various psychological stressors was randomized. Following the last day of the 40 -day exposure, the rats were euthanized and their testicular tissues were fixed in Bouin's solution. Moreover, a tissue processor, a microtome as well as hematoxylin, and eosin staining were used for tissue preparation. ResultsThe co-exposure to psychological and heat stress can cause negative testis histopathological changes including spermatid retention, degeneration of round spermatids and spermatocytes, degeneration and depletion of elongated Spermatid, Sertoli cell status, tubular degeneration/atrophy, Leydig cell atrophy, tubular dilatation, re-tubular dilation, and sclerosis status in a number of rats. Moreover, the histopathological changes were higher in the group exposed to heat stress compared with those exposed to psychological stress. ConclusionsAlthough exposure to either stressor alone can affect testicular tissue as part of the reproductive system, co-exposure to both stressors may result in an increased risk of adverse effects on testicular tissue.