Pubourethral and uterosacral male analogues suggest parallel male/female pelvic anatomy and symptom pathogenesis.

The thesis that functional/dysfunctional male/female pelvic floor anatomy are parallel, originated from two studies: a successful retropubic perineal male sling for post-prostatectomy stress urinary incontinence (SUI) and discovery of a male uterosacral ligament (USL) analogue, we named "prostatosacral ligament" (PSL). In 25 out of the studied 27 males (92.6%), it starts on both sides of the median sulcus of the prostate the ligament passes lateral to the rectum being fused with the lateral margin of the mesorectum before leaving it as it thins out to be attached posteriorly similar to the USL. The ultrasound data during straining in men and women showed the same three oppositely acting muscle vectors contracting around analogous ligaments, puboprostatic ligament (PPL) (male), and pubourethral ligament (PUL) (female). Further parallels were pubovesical ligaments (PVLs) and arc of Gilvernet as part of the continence and micturition mechanisms. Impressive evidence for parallel anatomy came from the successful cure of 22 males with post-prostatectomy SUI using a perineal retropubic tissue fixation system (TFS) minisling applied to the PPL using a similar methodology to that used in the female for PUL midurethral sling repair for cure of SUI. Laparoscopic evidence confirmed the prostate as a male analogue of the cervix, and PSLs as analogues of USLs: PSL origin from the prostate attached laterally to the mesorectum and inserted into the sacrum. Histologically, PSLs had identical features with USLs: collagen, elastin, smooth muscle, blood vessels and nerves. Virtually identical symptoms for "chronic prostatitis" (CP) and "posterior fornix syndrome" (PFS), such as chronic pelvic pain, overactive bladder (OAB), abnormal emptying, gave birth to the hypothesis, of a common pathogenesis for "CP" and "PFS", USL (or PSL) laxity. If this could be proven by "simulated operations", "CP", at least in theory, may be potentially correctible by PSL repair.

Standardization of honey as a tissue fixative for histopathology: A morphometric study.

Background: Tissue fixation is a crucial step to preserve the tissues in a life-like state with minimal disruption to its cellular and chemical composition for histopathological examination. The search for an effective alternate tissue fixative to the routinely used formaldehyde has gained interest as constant exposure to formaldehyde has proven to be toxic. Honey, an organic substance with high acidity and hygroscopic nature, exhibits tissue fixative properties and has been used in the present study. The present study aimed to standardize honey as a tissue fixative for histopathology by comparing it with formalin. Materials and Methods: In vitro study Oral tissue samples of goat were fixed in 10% honey and 10% formalin solution, respectively, for 24-48 h, followed by routine tissue processing and microscopic examination of 37 slides per group. 2200 epithelial cells (1100 per group) were selected for the computer-aided morphometric image analysis (Fiji-Image J) by three observers. Cell area (CA), cell perimeter (CP), nuclear area (NA), nuclear perimeter (NP), cytoplasmic area (Cyt A), and nuclear-cytoplasmic ratio were the parameters studied. Mann-Whitney U-test (STATA/IC version 16) for inter-group comparison was done and P < 0.05 was considered statistically significant. Results: The probability of epithelial cells in the honey-fixed group to have greater NA, NP, and N/C ratio was about 50%-60%. The probability of epithelial cells in formalin-fixed tissues to have greater CA, CP, and Cyt A was about 70%. Conclusion: Honey is a better nuclear fixative than formalin. Cytoplasmic shrinkage of epithelial cells should be taken into consideration while fixing tissues with honey.

Pragmatic guide to the macroscopic evaluation of breast specimens.

The pathological assessment of a breast surgical specimen starts with macroscopic evaluation, arguably one of the most critical steps, as only a small percentage of the tissue is examined microscopically. To properly evaluate and select tissue sections from breast specimens, it is essential to correlate radiological findings, prior biopsies, procedures and treatment with the gross findings. Owing to its fatty nature, breast tissue requires special attention for proper fixation to ensure appropriate microscopic evaluation and performance of ancillary studies. In addition, knowledge of the information necessary for patient management will ensure that these data are collected during the macroscopic evaluation, and appropriate sections are taken to obtain the information needed from the microscopic evaluation. Herein, we present a review of the macroscopic evaluation of different breast specimen types, including processing requirements, challenges and recommendations.

Macroscopy of specimens from the genitourinary system.

Macroscopic specimen examination is often critical for accurate histopathology reporting but has generally received insufficient attention and may be delegated to inexperienced staff with limited guidance and supervision. This review discusses issues around macroscopic examination of some common urological specimens; highlighting findings that are critical for patient management and others that are clinically irrelevant. Macroscopic findings are of limited value in completely submitted radical prostatectomy specimens but may be critical in orchidectomy specimens where identification of focal non-seminomatous components can significantly impact patient management. The maximum tumour dimension is often an important prognostic indicator, but specimen dimensions are generally of little clinical utility. Specimens should be carefully examined and judiciously sampled to identify clinically important focal abnormalities such as sarcomatoid change in a renal cell carcinoma and a minor non-seminomatous component in a predominant testicular seminoma. Meticulous macroscopic examination is key as less than 0.2% of the specimen (or macroscopically abnormal area) would be histologically examined even if the entire specimen/abnormal area is submitted for microscopic examination. Retroperitoneal pelvic lymph node dissection specimens for testicular cancer must be handled very differently from other lymph nodal block dissections. Current sampling protocols for transurethral resection of prostate specimens that are based on pre-MRI era data need to be reconsidered because they were specifically designed to detect occult prostate cancer, which would amount to histological cancer screening. Prostatic sampling of cystoprostatectomy specimens should be directed at accurately staging the known bladder cancer rather than detection of incidental prostate cancer.

Impact of the quality of resected thyroid cancer tissue sample on next-generation sequencing testing.

Activating rearranged during transfection (RET) proto-oncogene alterations can be identified using next-generation sequencing (NGS) of tumor DNA/RNA. We assessed factors associated with NGS (Oncomine Dx Target Test [ODxTT]) success for resected thyroid cancer (TC) specimens, including sample age, processing conditions, and DNA/RNA quality. TC samples were from three Japanese hospitals, with sample age <1-<10 years, fixative 10%/15% neutralized buffered formalin (NBF), and fixation time ≤48 h/>48 h-≤72 h. NGS success rate was defined as the percentage of samples returning validated NGS results (RET fusion-positive/negative [RNA] or RET mutation-positive/negative [DNA], detected using ODxTT). DNA/RNA quality was assessed with indexes based on electrophoresis (DNA/RNA integrity number, DV200 ) and quantitative polymerase chain reaction (DNA/RNA integrity score [ddCq/ΔCq]). NGS success rate (N = 202) was 90%/93% (DNA/RNA) overall, 98%-100% (DNA and RNA) for samples <3 years old, and 91% (DNA and RNA) for samples ≥3-<5 years old fixed in 10% NBF for ≤48 h. Multivariate logistic regression analysis identified ddCq and ΔCq as significant predictors of DNA and RNA NGS success rates, respectively. Quality assessment of nucleic acid extracted from archival tissue samples is important for achieving high NGS success rates in clinical practice, especially for samples ≥3 years old.

Morphological changes in striated muscle fibres caused by components of the Thiel embalming method.

BACKGROUND: Thiel-fixed body donors are highly valued for surgical training courses. The pronounced flexibility of Thiel-fixed tissue has been postulated to be caused by histologically visible fragmentation of striated muscle. The aim of this study was to analyse whether a specific ingredient, pH, decay, or autolysis could cause this fragmentation in order to modulate the Thiel solution to adapt specimen flexibility specifically to the needs of different courses. MATERIALS AND METHODS: Striated muscle of the mouse was fixed for different time periods in formalin, Thiel solution, and its individual ingredients, and analysed by light microscopy. Further, pH-values of Thiel solution and its ingredients were measured. In addition, unfixed muscle tissue was histologically analysed including Gram staining to investigate a relationship between autolysis, decomposition, and fragmentation. RESULTS: Muscle fixed with Thiel solution for 3 months was slightly more fragmentated than muscle fixed for 1 day. Fragmentation was more pronounced after 1 year of immersion. Three individual salt ingredients showed slight fragmentation. Decay and autolysis had no effect on fragmentation, which occurred regardless of the pH of all solutions. CONCLUSIONS: Fragmentation of Thiel-fixed muscle is dependent on fixation time and most likely occurs due to salts present in the Thiel solution. Adjustment of the salt composition in the Thiel solution with verification of the influence on the fixation effect, fragmentation and flexibility of the cadavers could be performed in further studies.

Macroscopic examination of pathology specimens: a critical reappraisal.

Meticulous macroscopic examination of specimens and tissue sampling are crucial for accurate histopathology reporting. However, macroscopy has generally received less attention than microscopy and may be delegated to relatively inexperienced practitioners with limited guidance and supervision. This introductory paper in the minisymposium, Macroscopy Under the Microscope, focuses on issues regarding macroscopic examination and tissue sampling that have been insufficiently addressed in the published literature. It highlights the importance of specimen examination and sampling, discusses some general principles, outlines challenges and suggests potential solutions. It is critical to get macroscopy right the first time as it may not be possible to rectify errors even with expert histological assessment or to retrospectively collect missing data after the specimen retention period. Dissectors must, therefore, receive adequate guidance and supervision until they are proficient in macroscopic specimen examination. We emphasise the importance of the clinical context, optimal specimen fixation, succinct and clinically relevant macroscopic descriptions, macrophotography and judicious tissue sampling. We note that current recommendations based on the number of blocks to be submitted per maximum tumour dimension are ambiguous as the amount of tissue submitted in a cassette is not standardised and it is unclear whether 'block' refers to a tissue block or a paraffin block. Concerns around potential oversampling of 'therapeutic' specimens that could result in overdiagnosis due to detection of incidentalomas are also discussed. We hope that the issues discussed in this paper will engender debate on this clinically critical aspect of pathology practice.

Challenges associated with vascular access through the common carotid artery during embalming procedures in humans: A technical note.

BACKGROUND: The teaching of anatomy is a key component in the training of physicians, and the foundation of this teaching is the human body, which must be properly prepared to be used as a teaching aid. Due to a lack of modern literature on this topic, we decided to write a technical note discussing access to the carotid artery. MATERIALS AND METHODS: We pre-qualified 43 donor bodies for the study. The bodies had to meet standards such as no signs of post-mortem decomposition, preservation of body integrity, and the absence of known infections. Carotid artery access was performed based on descriptions of the types of vascular access performed in surgery and our own observations. RESULTS: We consider carotid artery access to be a convenient option due to its ease of location. When performed correctly and with attention to the surrounding structures, it is relatively low in tissue trauma, which translates into a higher quality of preparation. Data analysis has revealed several factors that can have a significant impact on the success of the embalming procedure. CONCLUSIONS: Proper execution of minimally invasive access to the common carotid artery minimizes tissue damage and ensures a high success rate of the procedure. Knowledge of the types of vascular access is essential for preparing the highest quality specimens.

Preserving extracellular space for high-quality optical and ultrastructural studies of whole mammalian brains.

Analysis of brain structure, connectivity, and molecular diversity relies on effective tissue fixation. Conventional tissue fixation causes extracellular space (ECS) loss, complicating the segmentation of cellular objects from electron microscopy datasets. Previous techniques for preserving ECS in mammalian brains utilizing high-pressure perfusion can give inconsistent results owing to variations in the hydrostatic pressure within the vasculature. A more reliable fixation protocol that uniformly preserves the ECS throughout whole brains would greatly benefit a wide range of neuroscience studies. Here, we report a straightforward transcardial perfusion strategy that preserves ECS throughout the whole rodent brain. No special setup is needed besides sequential solution changes, and the protocol offers excellent reproducibility. In addition to better capturing tissue ultrastructure, preservation of ECS has many downstream advantages such as accelerating heavy-metal staining for electron microscopy, improving detergent-free immunohistochemistry for correlated light and electron microscopy, and facilitating lipid removal for tissue clearing.

Gelatin Zymography Can Be Performed on Fixed Brain Tissue.

Gelatin zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin zymography methods. (J Histochem Cytochem 71: 481-493, 2023).

Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation.

To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique requires neither separation of cells by enzymatic digestion of any kind nor cell-specific reporter genes, and it allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capturing of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue Preparation for Laser Capture Microdissection Basic Protocol 2: Tissue Sectioning Basic Protocol 3: Laser Capture Microdissection of Embedded Tissue Basic Protocol 4: RNA Extraction from Laser Capture Microdissection Samples.

Bone bridge technique for lateral meniscal allograft transplantation: no difference in clinical outcome compared to the soft tissue technique.

PURPOSE: There is considerable debate regarding the optimal method of fixation for lateral meniscus allograft transplantation (MAT), with bone bridge techniques technically harder but allowing maintenance of root attachments, while soft tissue techniques are potentially more challenging for healing. The aim of this study was to compare the clinical results of the bone bridge and soft tissue techniques for lateral MAT in terms of failure, re-operation rate, complications and patient reported outcomes. METHODS: Retrospective analysis of prospectively collected data for patients undergoing primary lateral MAT with a minimum of 12-month follow-up. Patients following surgery utilising the bone bridge technique (BB) were compared with historical control patients who underwent MAT with the soft tissue technique (ST). Outcome was assessed by failure rate, defined as removal or revision of the meniscus transplant, survivorship by Kaplan-Meir analysis, re-operation rates, and other adverse event. Patient-reported outcome measures (PROMs) were compared using data at the 2-year point or 1 year if not reached 2 years. RESULTS: One-hundred and twelve patients following lateral meniscal transplants were included, 31 in the BB group and 81 in the ST historical control group, with no differences in demographics between both groups. Median follow-up for the BB group was 18 (12-43) months compared to 46 (15-62) months for the ST group. There were 3 failures (9.6%) in the BB group v 2 (2.4%) in the ST group (n.s.) with a mean time to failure of 9 months in both groups. 9 patients (29%) required a re-operation (all cause) in the BB group v 24 patients (29.6%) in the ST group (n.s). There was no difference in complications between both groups. There was significant improvement (p < 0.0001) in all PROMs (Tegner, IKDC, KOOS and Lysholm) between baseline and 2-year follow-up for both groups but no between-group differences. CONCLUSION: Lateral MAT has a high success rate for symptomatic meniscal deficiency with significant benefits irrespective of the fixation technique. There is no advantage in performing the more technically demanding BB technique over ST fixation. LEVEL OF EVIDENCE: Level 2.

The impact of impaired tissue fixation in resected non-small-cell lung cancer on protein deterioration and DNA degradation.

OBJECTIVES: The objective is to assess the impact of the quality of tissue fixation in surgical pathology on immunohistochemical (IHC) staining and DNA degradation. MATERIALS AND METHODS: Twenty-five non-small cell lung cancer (NSCLC) resection specimens were analyzed. After resection, all tumors were processed according to the protocols in our center. In haematoxylin and eosin (H&E) stained tissue slides, adequately- and inadequately fixed tumor areas were microscopically demarcated, based on basement membrane detachment. In 10 IHC stains ALK (clone 5A4), PD-L (clone 22C3), CAM5.2, CK7, c-Met, KER-MNF116, NapsinA, p40, ROS1, TTF1) the immunoreactivity in H-scores was determined in adequately- and inadequately fixed, and necrotic tumor areas. From the same areas DNA was isolated, and DNA fragmentation in base pairs (bp) was measured. RESULTS: H-scores were significantly higher in H&E adequately fixed tumor areas in IHC stains KER-MNF116 (H-score 256 vs 15, p=0.001) and p40 (H-score 293 vs 248, p=0.028). All other stains showed a trend towards higher immunoreactivity in H&E adequately fixed areas. Independent of H&E adequatelty- or inadequately fixed areas, all IHC stains showed significant different IHC staining intensity within tumors, suggesting heterogeneous immunoreactivity (H-scores: PD-L1 123 vs 6, p = 0.001; CAM5.2 242 vs 101, p=<0.001; CK7 242 vs 128, p=<0.001; c-MET 99 vs 20, p=<0.001; KER-MNF116 281 vs 120, p=<0.001; Napsin A 268 vs 130, p = 0.005; p40 292 vs 166, p = 0.008; TTF1 199 vs 63, p=<0.001). DNA fragments rarely exceeded a length of 300 bp, independent of adequate fixation. However, DNA fragments of 300 and 400 bp had higher concentrations in tumors with short fixation delay (<6 h vs >16 h) and short fixation time (<24 h vs >24 h). CONCLUSIONS: Impaired tissue fixation of resected lung tumors results in decreased IHC staining intensity in some parts of the tumor. This may impact the reliability of IHC analysis.

Tissue Fixation with a Formic Acid-Deprived Formalin Better Preserves DNA Integrity over Time.

INTRODUCTION: Optimization of pre-analytic procedures and tissue processing is a basic requirement for reliable and reproducible data to be obtained. Tissue fixation in formalin represents the extensively favored method for surgical tissue specimen processing in diagnostic pathology; however, formalin fixation exerts a blasting effect on DNA and RNA. METHODS: A formic acid-deprived formaldehyde solution was prepared by removing acids with an ion-exchange basic resin and the concentrated, acid-deprived formaldehyde (ADF) solution was employed to prepare a 4% ADF solution in 0.1 M phosphate buffer, pH 7.2-7.4. Human (n = 27) and mouse (n = 20) tissues were fixed in parallel and similar conditions in either ADF or neutral buffered formalin (NBF). DNAs and RNAs were extracted, and fragmentation analyses were performed. RESULTS: Besides no significant differences in terms of extraction yield and absorbance ratio, ADF fixation reduced DNA fragmentation, i.e., the largest fragments (>5,000 bp) were significantly more prevalent in the DNAs purified from ADF-fixed tissues (p < 0.001 in both cohorts). Moreover, we observed that DNA preservation is more stable in ADF-fixed tissue compared to NBF-fixed tissues. CONCLUSION: Although DNA fragmentation in FFPE tissues is a multifactor process, we showed that the removal of formic acid is responsible for a significant improvement in DNA preservation.

Protocol for the Systematic Fixation, Circuit-Based Sampling, and Qualitative and Quantitative Neuropathological Analysis of Human Brain Tissue.

Human brain tissue has long been a critical resource for neuroanatomy and neuropathology, but with the advent of advanced imaging and molecular sequencing techniques, it has become possible to use human brain tissue to study, in great detail, the structural, molecular, and even functional underpinnings of human brain disease. In the century following the first description of Alzheimer's disease (AD), numerous technological advances applied to human tissue have enabled novel diagnostic approaches using diverse physical and molecular biomarkers, and many drug therapies have been tested in clinical trials (Schachter and Davis, Dialogues Clin Neurosci 2:91-100, 2000). The methods for brain procurement and tissue stabilization have remained somewhat consistently focused on formalin fixation and freezing. Although these methods have enabled research protocols of multiple modalities, new, more advanced technologies demand improved methodologies for the procurement, characterization, stabilization, and preparation of both normal and diseased human brain tissues. Here, we describe our current protocols for the procurement and characterization of fixed brain tissue, to enable systematic and precisely targeted diagnoses, and describe the novel, quantitative molecular, and neuroanatomical studies that broadly expand the use of formalin-fixed, paraffin-embedded (FFPE) tissue that will further our understanding of the mechanisms underlying human neuropathologies.

A suturable biohydrogel with mechanical matched property based on coating chitosan and polyethylene glycol shell for tissue patching.

The mechanical mismatch between soft hydrated tissues and sutures has become a common negative impact on wound healing process. A novel method of coating multilayer polymer shells is thus reported to improve the mechanical performance of hydrogel sutures. It is suitable for tissue patching and shows advantages of convenient, efficient, and biosafety. Specifically, a precursor hydrogel (Cu@CMC) consisted of carboxymethyl chitosan and copper modified by carbon dots was used as the inner sheath, and then bonding the precursor hydrogel sheath with toughening polyethylene glycol network by anchoring sites composited from rigid chitosan shell integrated a whole structure. Subsequently, the whole system was soaked with EtOH, and rapid dehydration of EtOH was used to accelerate the entanglement process between the two coatings by constricting the molecular chains. Finally, an ideal suture (Cu-fiber) with both toughness and rigidness was obtained. The data showed that the tensile strength and biosafety of the hydrogel sutures prepared by the new strategy were significantly improved, and the skin, liver and vessel of rodents can be sutured without secondary damage. Moreover, it can inhibit inflammation response and promote the healing process of skin wound, indicating that the Cu-fiber will become a great candidate for tissue patching.

Thirty years of Thiel embalming-A systematic review on its utility in medical research.

In 1992, Walter Thiel described and embalming method that rendered "lifelike" tissues. Over the last 30 years, the Thiel method has been introduced worldwide for medical training and scientific purposes. This review examines research which can be linked to the use of Thiel embalming. A systematic review was performed to identify articles published in the following categories: research content, disciplines involved, sources and quantities of tissues deployed, and changes in research scope related to changes in the chemical composition of Thiel embalming. Four-hundred twenty-four publications were included. A number of adaptations to the original Thiel protocol were found, aiming to provide suitable tissue-substitutes in the development of emerging medical technologies or procedures. Musculoskeletal surgery, anesthesia and intensive care were the most common disciplines that used Thiel embalmed tissues for research. Anatomy and biomechanics played a lesser role. An increase over time was observed in research outputs related to the Thiel method, while the number of specimens used per study decreased. The main centers using Thiel embalming were in Graz, Dundee, Sapporo, Bern, Zurich and Ghent, which jointly accounted for more than 54% of all research conducted using this method. Following three decades of use, the Thiel method has evolved into being a well-established embalming technique for research purposes. Its future is challenged by the demanding requirements on both technical facilities and personnel, limitations of certain chemicals for use as fixatives, costs, and questions as to how "lifelike" the embalmed-tissues are from an objective standpoint, all of which warrants future investigations.

Biomechanical Comparison of Three Suspensory Techniques for all Soft Tissue Central Quadriceps Tendon Graft Fixation.

Purpose: The purpose of this study was to evaluate three different methods of attachment of continuous loop suspensory cortical preparation of all soft tissue central quad tendon grafts compared to a bone block control for anterior cruciate ligament reconstruction on construct displacement and load to failure. Methods: Thirty-two cadaveric central quadriceps tendon (CQT) specimens were harvested, using three clinical techniques for graft fixation: cortical button alone (BTB EB), BTB cortical button with rip-stop suture (BTB RS), and continuous loop cortical button (BTB CL). A control group was also included that consisted of a bone block secured within testing clamps (BTB CON). Specimens were preloaded to 150 N. Tendons were then cyclically loaded between 50 N and 250 N for 1,000 cycles at .5 Hz. Displacement was measured at the point of fixation of the CQT after the 150 N preload, 250 N initial load and every 100th cycle. The specimens were loaded to failure after 1,000 cycles. Results: There was a significant increase in displacement from .32 ± .56 mm for the BTB CON to 1.91 ± 1.13 mm for the BTB RS (P = .014) and 3.85 ± 2.32 mm for the BTB CL condition (P = .023). There was no significant increase in displacement for BTB EB (P = .182). Failure occurred for all of the BTB CL and 62.5% of the BTB EB specimens within the first 50 cycles. Twenty-five percent of the BTB CON specimens and 12.5% BTB RS failed at ∼400 and 500 cycles, respectively. Similar failure loads were observed for the BTB CON and the BTB RS (446.4 ± 151.46 N vs 505.74 ± 131.41 N; P = .99) Failure testing was not feasible for the BTB CL and BTB EB preparation methods. Conclusion: In response to cyclic loading, the three all-soft tissue suspensory conditions experienced significantly greater displacement compared to the bone block controls. None of the soft-tissue conditions appeared superior when compared to each other. Clinical Relevance: It remains unknown which method of soft-tissue suspensory provides optimal fixation. As these autografts become more common, it is essential to evaluate which fixation methods provide superior outcomes.

Glutaraldehyde fixation of venous valve tissue: A benchmark for alternative fixation methods.

OBJECTIVE: Bioprosthetic venous valves have yet to achieve long-term patency due to issues with calcification following implantation that is influenced by current xenograft fixation methods, most notably glutaraldehyde. The goal of this study was to investigate the effects of glutaraldehyde fixation on the functional properties of venous tissue to establish a benchmark for the evaluation of alternative fixation methods. METHODS: The degree of crosslinking was evaluated by determining shrink temperature and the stability of tissue with pronase and collagenase digestion. RESULTS: Glutaraldehyde fixation of venous tissue was confirmed by a significant difference in the shrink temperature between fresh and glutaraldehyde treated samples. Significant differences in the amount of tissue remaining following digestion were observed for venous versus cardiac tissue. CONCLUSIONS: This study demonstrates the importance of tissue-specific evaluation in the development of alternative xenograft fixation methods to improve outcomes with bioprosthetic venous valves.

Perfusion with 10% neutral-buffered formalin is equivalent to 4% paraformaldehyde for histopathology and immunohistochemistry in a mouse model of experimental autoimmune encephalomyelitis.

Intravascular (IV) perfusion of tissue fixative is commonly used in the field of neuroscience as the central nervous system tissues are exquisitely sensitive to handling and fixation artifacts which can affect downstream microscopic analysis. Both 10% neutral-buffered formalin (NBF) and 4% paraformaldehyde (PFA) are used, although IV perfusion with PFA is most commonly referenced. The study objective was to compare the severity of handling and fixation artifacts, semiquantitative scores of inflammatory and neurodegenerative changes, and quantitative immunohistochemistry following terminal IV perfusion of mice with either 10% NBF or 4% PFA in a model of experimental autoimmune encephalitis (EAE). The study included 24 mice; 12 were control animals not immunized and an additional 12 were immunized with PLP139-151 subcutaneously, harvested at day 20, and fixed in the same fashion. Equal numbers (4 per group) were perfused with 10% NBF or 4% PFA, and 4 were immersion-fixed in 10% NBF. NBF-perfused mice had less severe dark neuron artifact than PFA-perfused mice (P < .001). Immersion-fixed animals had significantly higher scores for oligodendrocyte halos, dark neuron artifact, and perivascular clefts than perfusion-fixed animals. Histopathology scores in EAE mice for inflammation, demyelination, and necrosis did not differ among fixation methods. Also, no significant differences in quantitative immunohistochemistry for CD3 and Iba-1 were observed in immunized animals regardless of the method of fixation. These findings indicate that IV perfusion of mice with 10% NBF and 4% PFA are similar and adequate fixation techniques in this model.