Tissue Fixation Research Articles

Anterior cruciate ligament reconstruction in a translational model in sheep using biointegrative mineral fiber reinforced screws

Anterior cruciate ligament reconstruction (ACLR) is one of the more common procedures performed worldwide and perhaps the most widely studied construct in orthopedic literature. Interference screws are reliable and frequently used for ligament reconstruction, providing rigid fixation and facilitates graft incorporation allowing for the physiologic loads of early rehabilitation. The purpose of this study was to determine the bio-integration profile and quality of soft tissue graft when using mineral fiber-reinforced screws in an ACLR interference model. Nine sheep underwent ACLR using harvested autologous tendon graft fixated with 4.75 mm screws made of continuous mineral fibers. Histopathology and imaging evaluation at 28, 52, 104, 132-weeks (W) demonstrated mesenchymal tissue ingrowth into implant wall at 28 W, which increased at 52 W and peaked at 104 W. At 132 W, implants fully replaced by newly remodeled bone. Graft cellularity was evident at 28 W and continued to increase through 132 W as the tendon ossified at sites of bone contact. Pro-healing M2-macrophages and giant cells remained infrequent, with minor increases between 52 W and 104 W, attributed to expected phagocytic response. Pro-inflammatory cells (i.e., M1-macrophages, polymorphonuclears) were absent through the entire study course. In conclusion, bio-integrative screws provide secure soft tissue fixation with replacement by bone demonstrating graft cellularization over time.

Relationship between regional volume changes and water diffusion in fixed marmoset brains: an in vivo and ex vivo comparison

Ex vivo studies of the brain are often employed as experimental systems in neuroscience. In general, brains for ex vivo MRI studies are usually fixed with paraformaldehyde to preserve molecular structure and prevent tissue destruction during long-term storage. As a result, fixing brain tissue causes microstructural changes and a decrease in brain volume. Therefore, the purpose of this study was to investigate the regional effect of brain volume and microstructural changes on the restricted diffusion of water molecules in the common marmoset brain using in vivo and ex vivo brains from the same individual. We used 9.4T magnetic resonance imaging and also compared the T2-weighted images and diffusion-weighted imaging (DWI) data between in vivo and ex vivo brains to investigate changes in brain volume and diffusion of water molecules in 12 common marmosets. We compared fractional anisotropy, mean diffusivity, AD (axial diffusivity), and radial diffusivity values in white matter and gray matter between in vivo and ex vivo brains. We observed that AD showed the strongest correlation with regional volume changes in gray matter. The results showed a strong correlation between AD and changes in brain volume. By comparing the in vivo and ex vivo brains of the same individual, we identified significant correlations between the local effects of perfusion fixation on microstructural and volumetric changes of the brain and alterations in the restricted diffusion of water molecules within the brain. These findings provide valuable insights into the complex relationships between tissue fixation, brain structure, and water diffusion properties in the marmoset brain.

A powerful and versatile new fixation protocol for immunostaining and in situ hybridization that preserves delicate tissues

BackgroundUnderstanding how genes function to heal wounds and restore lost tissue is essential for studying regeneration. Whole-mount in situ hybridization (WISH) is a powerful and widely used technique to visualize the expression patterns of genes in different biological systems. Yet, existing methods to permeabilize samples for WISH can damage or destroy fragile regenerating tissues, thereby preventing such experiments.ResultsHere, we describe a new protocol for in situ hybridization (ISH) and immunostaining in the highly regenerative planarian Schmidtea mediterranea. This new Nitric Acid/Formic Acid (NAFA) protocol is compatible with both the assays and prevents degradation of the epidermis and regeneration blastema. The NAFA protocol achieves this without the use of proteinase K digestion which likely leads to better preservation of antigen epitopes. We show that the NAFA protocol successfully permits development of chromogenic and fluorescent signals in situ, while preserving the anatomy of the animal. Furthermore, the immunostaining of different proteins was compatible with the NAFA protocol following fluorescent in situ hybridization. Additionally, the tissue fixation protocol was easily adapted for regenerating killifish tail fin, which yielded better ISH signal with minimal background.ConclusionsThus, the NAFA protocol robustly preserves the delicate wounded tissues while also facilitating probe and antibody penetration into internal tissues. Furthermore, the fixation protocol is compatible for WISH on regenerating teleost fins suggesting that it will be a valuable technique for studying the processes of wounding response and regeneration in multiple species.

Metatranscriptomic analysis to reveal the coupling between nitrogen fixation and CH4 oxidation in root tissues of Phragmites australis

Metatranscriptomic analysis to reveal the coupling between nitrogen fixation and CH4 oxidation in root tissues of Phragmites australis

Investigating microstructural changes between in vivo and perfused ex vivo marmoset brains using oscillating gradient and b-tensor encoded diffusion MRI at 9.4 T.

To investigate microstructural alterations induced by perfusion fixation in brain tissues using advanced diffusion MRI techniques and estimate their potential impact on the application of ex vivo models to in vivo microstructure. We used oscillating gradient spin echo (OGSE) and b-tensor encoding diffusion MRI to examine in vivo and ex vivo microstructural differences in the marmoset brain. OGSE was used to shorten effective diffusion times, whereas b-tensor encoding allowed for the differentiation of isotropic and anisotropic kurtosis. Additionally, we performed Monte Carlo simulations to estimate the potential microstructural changes in the tissues. We report large changes (˜50%-60%) in kurtosis frequency dispersion (OGSE) and in both anisotropic and isotropic kurtosis (b-tensor encoding) after perfusion fixation. Structural MRI showed an average volume reduction of about 10%. Monte Carlo simulations indicated that these alterations could likely be attributed to extracellular fluid loss possibly combined with axon beading and increased dot compartment signal fraction. Little evidence was observed for reductions in axonal caliber. Our findings shed light on advanced MRI parameter changes that are induced by perfusion fixation and potential microstructural sources for these changes. This work also suggests that caution should be exercised when applying ex vivo models to infer in vivo tissue microstructure, as significant differences may arise.

Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains.

For obtaining insights into gene networks during plant reproductive development, having transcriptomes of specific cells from developmental stages as starting points is very useful. During development, there is a balance between cell proliferation and differentiation, and many cell and tissue types are formed. While there is a wealth of transcriptome data available, it is mostly at the organ level and not at specific cell or tissue type level. Therefore, methods to isolate specific cell and tissue types are needed. One method is fluorescent activated cell sorting (FACS), but it has limitations such as requiring marker lines and protoplasting. Recently, single-cell/nuclei isolation methods have been developed; however, a minimum amount of genetic information (marker genes) is needed to annotate/predict the resulting cell clusters in these experiments. Another technique that has been known for some time is laser-assisted microdissection (LAM), where specific cells are microdissected and collected using a laser mounted on a microscope platform. This technique has advantages over the others because no fluorescent marker lines must be made, no marker genes must be known, and no protoplasting must be done. The LAM technique consists in tissue fixation, tissue embedding and sectioning using a microtome, microdissection and collection of the cells of interest on the microscope, and finally RNA extraction, library preparation, and RNA sequencing. In this protocol, we implement the use of normal slides instead of the membrane slides commonly used for LAM. We applied this protocol to obtain the transcriptomes of specific tissues during the development of the gynoecium of Arabidopsis. Key features • Laser-assisted microdissection (LAM) allows the isolation of specific cells or tissues. • Normal slides can be used for LAM. • It allows the identification of the transcriptional profiles of specific tissues of the Arabidopsis gynoecium.

Histological methods for plant tissues

ABSTRACT Although many of the structures and organelles of vegetative cells are comparable to those of animal tissues, significant differences between the two kingdoms require modifications in histological techniques for both tissue processing steps and histochemical staining techniques. The authors investigated the challenges of working with plant tissues by collecting various flora to represent the four main plant organs: leaf, stem, root, and flower/fruit. Triplicate samples for each specimen were placed into formalin for paraffin embedding, placed into formalin for later frozen sections, and used fresh to undergo immediate frozen sectioning. Frozen sections of plant tissues were more difficult to obtain than formalin-fixed paraffin-embedded (FFPE) sections, exhibited tissue loss during staining, and were inferior morphologically to FFPE sections. Although, historically, plant tissue fixation and processing has employed several different reagents compared with those used in animal tissue processing and took significantly longer times, the current investigation determined reagents and protocols from a modern histology laboratory which processes mammalian tissues can be applied to plant tissue processing with only slight modifications in respect to reagent timing. Additionally, staining techniques were compared and while it is well known that plant cell walls stain well with safranin O, the current investigation determined the uptake of safranin O can be accelerated by incubating at 60°C.

Evaluating the effectiveness of various agents as interim preservative media for biopsy specimens: An experimental in vitro study

In the field of medicine, diagnosing diseases involves various steps, procedures, and protocols. Histopathological examination remains the gold standard for a definitive and accurate diagnosis. This process relies on an initial step of tissue fixation.Formaldehyde is a traditional fixative material and is typically used as 10% or 4% buffered solutions. However, significant health risks are associated with formaldehyde exposure.Our paper aimed to evaluate and compare variable media as a preservative agent. The study included eight groups as follows: (1) normal saline 0.9%; (2) local anesthesia with epinephrine (2% Lidocaine HCl/ 1:100,000 epi); (3) local anesthesia without epinephrine (3% Mepivacaine HCl/ plain); (4) distilled water; (5) Saliva collected from healthy male participants; (6) Milk (Almarai- full-fat fresh milk); (7) Propolis; and (8) l0% formalin. Three tissue samples from each group were fixed in 10% formalin for 24 h after being preserved for 6 h at ambient temperature in their respective media. All specimens were stained with Hematoxylin and Eosin.The following histological structures were assessed: (1) superficial 1/3 of the epithelium, (2) middle 1/3 of the epithelium, (3) lower 1/3 of the epithelium, (4) basement membrane/rete ridges, (5) fibrous connective tissue, (6) blood vessels/endothelial cells, (7) skeletal muscle, (8) adipose tissue, and (9) nerve bundles. A numerical rating system was employed to assess these components, with scores ranging from 1 to 4.Our findings suggest propolis has promising potential as a tissue fixative. This study highlighted the effectiveness of propolis as a preservative and fixative solution, which warrants further investigation.

Immunohistochemistry in Cancer Diagnosis: A Review

Abstract Immunohistochemistry in cancer diagnosis holds great potential to improve patient’s outcomes through enhanced diagnostic accuracy and more effective treatment strategies. Antigen-antibody interactions is one of its main objectives that utilizes the specific binding of antibodies to cellular antigens allowing for the precise identification and localization of specific proteins within tissue samples. However, there exist practical hitches in the form of tissue fixation, epitope retrieval, and antibody quality. It is crucial to continue streamlining the techniques and scoring systems to guarantee their reliability in various contexts. Understanding the interactions is pivotal for the accurate diagnosis, prognosis, and future perspectives. As technology continues to advance and our understanding of cancer biology deepens, immunohistochemistry will remain an invaluable tool for precision in cancer diagnosis.

A cross-sectional study to correlate nuclear fixative properties of formal saline and clarke’s fluid for histomorphology evaluation of human tissues

Abstract Background: Formal saline is an extensively used cheapest and easily available tissue fixative. However, formalin has been proven to have carcinogenic effects. Clarke’s fluid is an alternative to formal saline and is effective in maintaining the morphological and cellular architecture. Objectives: The objective of the study was to compare the nuclear fixative properties of Clarke’s fluid and formal saline. Materials and Methods: The fresh human tissues of thin skin, large-sized arteries, lymph nodes, spleen, and ileum were obtained from the fresh cadaver. The tissues were fixed in two different fixatives namely 10% formalin and Clarke’s fluid. Then, the tissues were histologically processed. The hematoxylin and eosin-stained serial sections of tissues were graded based on the ease of microtomy, cytoplasmic, and nuclear staining scores. Results: The nuclear properties of all the tissues immersed in Clarke’s fluid showed a median score of 3 and Grade 2 for formal saline; cytoplasmic properties for spleen and skin were median scores of Grade 3 for Clarke’s fluid. The artery was graded 3 for both the fixatives. The Kruskal–Wallis test when applied to nuclear properties, cytoplasmic adequacy, and tissue shrinkage for tissues stained with Clarke’s fluid was statistically significant (P < 0.01). This inferred that Clarke’s fluid is a better nuclear fixative than formal saline. Conclusion: The imminent risks associated with formal saline paved us to explore substitute fixatives, which could provide better results and safety for laboratory workers. Clarke’s fluid is an excellent substitute for formal saline as a nuclear fixative.

Effectiveness of May Grunwald Giemsa staining on tissues fixed with sugar cane molasses

Formaldehyde is a reactive chemical that reacts randomly with various cellular elements. This substance, which is also found in the natural structure of the organism, is used in many areas, from the industry to household materials, from the production of coatings in dentistry to the fixation of cadavers in laboratories. Formaldehyde is commonly used in laboratories for fixation of tissues. In this study, the staining properties of tissues fixed with low-cost sugar cane molasses, which is one of the natural sugars and organically produced in Turkey, shown by May Grunwald Giemsa staining method were compared with those of tissues fixed with buffered formol-saline. As a result, intense maceration was observed in the skin and small intestines. Considering the chromatin distribution, nucleus separation and cytoplasm staining in other tissues (liver, spleen, kidney, testis, large intestine, brain, cerebellum and lung), tissues fixed with sugarcane molasses showed similar properties to tissues fixed with buffered formal-saline.

Different characteristics between acute and delayed proximal junctional failure in elderly patients undergoing corrective surgery for adult spinal deformity: comparative analysis of risk factor, failure mode, and clinical consequences

Background contextWhile numerous studies have been conducted on proximal junctional failure (PJF), the clinical significance of acute and delayed PJF remains poorly understood. PurposeThe primary object of this study is to investigate the risk factors separately for acute and delayed PJF. Secondly, we aim to assess the incidence of each failure mode and their clinical consequences in relation to acute and delayed PJF. Study design/settingRetrospective comparative study. Patient samplePatients aged ≥60 years who underwent deformity correction with ≥5-level fusion to sacrum. Outcome measuresRisk factor, failure modes, and patient-reported outcome measure (PROM) MethodsAcute PJF is defined as PJF occurring within 6 months, while delayed PJF occurring after 6 months. Risk factors were analyzed by comparing various clinical and radiographic parameters among three groups: no, acute, and delayed PJF groups. The failure modes, including soft tissue failure, vertebral fracture, fixation failure, and myelopathy, were compared among these groups. The clinical subsequences after PJF development were evaluated by assessing the change in proximal junctional angle (PJA), revision rate, and patient-reported outcome measure (PROM). ResultsA study cohort of 363 patients was included in the analysis. Among them, 156 patients experienced PJF, with 87 patients (55.8%) in the acute PJF group and 69 patients (44.2%) in the delayed PJF group. Multivariate analyses showed that older age (Odds ratio [OR] = 1.057, 95% confidence interval [CI] = 1.002 – 1.118), osteoporosis (OR=2.149, 95% CI = 1.074 – 4.300), high American Society of Anesthesiology ASA score (OR=2.150, 95% CI = 1.089 – 4.245), and overcorrection relative to the age-adjusted pelvic incidence - lumbar lordosis target (OR=4.031, 95% CI = 1.962 – 8.280) were identified as risk factors for the development of acute PJF. On the other hand, a high body mass index (OR=1.150, 95% CI = 1.049 – 1.251) and an uppermost instrumented vertebra located at ≤ T10 (OR=2.267, 95% CI = 1.205 – 4.268) were found to be associated with delayed occurrence of PJF. No radiographic parameters were found to be related to the development of delayed PJF. In terms of failure modes, vertebral fracture and fixation failure were more commonly observed in acute PJF, while soft tissue failure and myelopathy were more predominant in delayed PJF. The clinical course was more aggressive in the acute PJF group compared to the delayed PJF group, as evidenced by a greater increase in PJA, a higher revision rate, and worse PROM. ConclusionsThis study demonstrated different risk factors between the acute and delayed PJF. It was found that overcorrection relative to the age-adjusted PI-LL target increased the risk of acute PJF, but had no impact on the development of delayed PJF. Therefore, a different surgical strategy needs to be established to mitigate both acute and delayed PJF.

Deep Learning Artificial Intelligence Predicts Homologous Recombination Deficiency and Platinum Response From Histologic Slides.

Cancers with homologous recombination deficiency (HRD) can benefit from platinum salts and poly(ADP-ribose) polymerase inhibitors. Standard diagnostic tests for detecting HRD require molecular profiling, which is not universally available. We trained DeepHRD, a deep learning platform for predicting HRD from hematoxylin and eosin (H&E)-stained histopathological slides, using primary breast (n = 1,008) and ovarian (n = 459) cancers from The Cancer Genome Atlas (TCGA). DeepHRD was compared with four standard HRD molecular tests using breast (n = 349) and ovarian (n = 141) cancers from multiple independent data sets, including platinum-treated clinical cohorts with RECIST progression-free survival (PFS), complete response (CR), and overall survival (OS) endpoints. DeepHRD predicted HRD from held-out H&E-stained breast cancer slides in TCGA with an AUC of 0.81 (95% CI, 0.77 to 0.85). This performance was confirmed in two independent primary breast cancer cohorts (AUC, 0.76 [95% CI, 0.71 to 0.82]). In an external platinum-treated metastatic breast cancer cohort, samples predicted as HRD had higher complete CR (AUC, 0.76 [95% CI, 0.54 to 0.93]) with 3.7-fold increase in median PFS (14.4 v 3.9 months; P = .0019) and hazard ratio (HR) of 0.45 (P = .0047). There were no significant differences in nonplatinum treatment outcome by predicted HRD status in three breast cancer cohorts, including CR (AUC, 0.39) and PFS (HR, 0.98, P = .95) in taxane-treated metastatic breast cancer. Through transfer learning to high-grade serous ovarian cancer, DeepHRD-predicted HRD samples had better OS after first-line (HR, 0.46; P = .030) and neoadjuvant (HR, 0.49; P = .015) platinum therapy in two cohorts. DeepHRD can predict HRD in breast and ovarian cancers directly from routine H&E slides across multiple external cohorts, slide scanners, and tissue fixation variables. When compared with molecular testing, DeepHRD classified 1.8- to 3.1-fold more patients with HRD, which exhibited better OS in high-grade serous ovarian cancer and platinum-specific PFS in metastatic breast cancer.

Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue.

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ileum. Each anatomical region of the intestine has a unique function that is reflected by differences in cellular structure. Investigating changes in the intestine requires an in-depth analysis of different tissue regions and cellular alterations. To study the intestine and visualize large pieces of tissue, researchers commonly use a technique known as intestinal Swiss rolls. In this technique, the intestine is divided into each anatomical region and fixed in a flat orientation. Then, the tissue is carefully rolled and processed for paraffin embedding. Proper tissue fixation and orientation is an often-overlooked laboratory technique but is critically important for downstream analysis. Additionally, improper Swiss rolling of intestinal tissue can damage the fragile intestinal epithelium, leading to poor tissue quality for immunostaining. Ensuring well-fixed and properly oriented tissue with intact cellular structures is a crucial step that ensures optimal visualization of intestinal cells. We present a cost-effective and simple method for making Swiss rolls to include all sections of the intestine in a single paraffin-embedded block. We also describe optimized immunofluorescence staining of intestinal tissue to study various aspects of the intestinal epithelium. The following protocol provides researchers with a comprehensive guide to obtaining high-quality immunofluorescence images through intestinal tissue fixation, Swiss-roll technique, and immunostaining. Employing these refined approaches preserves the intricate morphology of the intestinal epithelium and fosters a deeper understanding of intestinal physiology and pathobiology.

P-075 A new clinically relevant classification of seminiferous tubules in Klinefelter Syndrome patients based on the karyotype of the Sertoli cells

Abstract Study question Can a new seminiferous tubule classification be made that indicates the chance of sperm retrieval by TESE in Klinefelter Syndrome (KS) patients? Summary answer Spermatogonia are only present in tubules with euploid Sertoli cells. Classification of tubules based on Sertoli cell ploidy may predict sperm retrieval success with TESE. What is known already KS patients can have children thanks to focal spermatogenesis. However, this can only happen if sperm is produced. Histological classification of seminiferous tubules in these patients has been made based on the maturity of the Sertoli cells (type A and type B seminiferous tubules). However, so far there is no classification predicting the presence of germ cells. In case two X chromosomes are present, one is inactivated. The histone-3-lysine-27-tri-methylation (H3K27me3) protein is involved in X chromosome inactivation and its immunohistochemical staining may be used for a new seminiferous tubule classification based on the karyotype of Sertoli cells. Study design, size, duration Inactive X (Xi) chromosomes were immunohistochemically stained with H3K27me3 and seminiferous tubules were classified as Xi+ tubules (presence of inactive X in Sertoli cells) or Xi- tubules (absence of inactive X in Sertoli cells). Testicular biopsies were used from 17 non-mosaic KS adult patients. Participants/materials, setting, methods After tissue fixation, processing, and paraffin embedding, sections were cut at 5 µm. Immunohistochemical staining was applied with antibodies H3K27me3 (for inactive X chromosome), MAGE-A4 (for spermatogonia), and SOX9 (for Sertoli cells). Seminiferous tubules in testicular sections were classified as Xi+ or Xi- tubules and analyzed for the presence of spermatogonia or focal spermatogenesis. X chromosome number in spermatogonia and Sertoli cells was confirmed by the FISH method using chromosome 18, Y, and X probes. Main results and the role of chance A total of 311 (18±16) seminiferous tubule sections were analyzed in testicular tissue of 17 KS men. Of the 311 tubules analyzed, 253 (14±16) of the tubules were Xi- tubules and 58 (3±4) were Xi+ tubules. Spermatogonia were present in 41 of the Xi- tubules. As an exception, only one patient had spermatogonia in two Xi+ tubules, but these tubules were immature and spermatogenesis was not observed. Ongoing spermatogenesis was observed in two of Xi- tubules in one of the patients. Sixteen of the patients had both Xi- tubules and Xi+ tubules in their testicular sections. In 13 of these 16 patients with Xi- tubules, sperm was either obtained as a result of TESE or spermatogonia were observed in the Xi- tubules. Limitations, reasons for caution In Klinefelter patients, there are few seminiferous tubules due to widespread testicular fibrosis and degeneration of seminiferous tubules. Studies including more patient samples are needed for more definitive and comprehensive conclusions. Wider implications of the findings Spermatogonia are only present in tubules with euploid Sertoli cells. Therefore, we hypothesize that focal spermatogenesis only can occur in tubules with euploid Sertoli cells. Xi+ and Xi- seminiferous tubule classification based on H3K27me3 staining may thus be used to predict sperm production in KS patients. Trial registration number Not applicable

An optimized workflow for transcriptomic analysis from archival paraformaldehyde-fixed retinal tissues collected by laser capture microdissection

RNA sequencing (RNA-seq) coupled with laser capture microdissection (LCM) is a powerful tool for transcriptomic analysis in unfixed fresh-frozen tissues. Fixation of ocular tissues for immunohistochemistry commonly involves the use of paraformaldehyde (PFA) followed by embedding in Optimal Cutting Temperature (OCT) medium for long-term cryopreservation. However, the quality of RNA derived from such archival PFA-fixed/OCT-embedded samples is often compromised, limiting its suitability for transcriptomic studies. In this study, we aimed to develop a methodology to extract high-quality RNA from PFA-fixed canine eyes by utilizing LCM to isolate retinal tissue. We demonstrate the efficacy of an optimized LCM and RNA purification protocol for transcriptomic profiling of PFA-fixed retinal specimens. We compared four pairs of canine retinal tissues, where one eye was subjected to PFA-fixation prior to OCT embedding, while the contralateral eye was embedded fresh frozen (FF) in OCT without fixation. Since the RNA obtained from PFA-fixed retinas were contaminated with genomic DNA, we employed two rounds of DNase I treatment to obtain RNA suitable for RNA-seq. Notably, the quality of sequencing reads and gene sets identified from both PFA-fixed and FF tissues were nearly identical. In summary, our study introduces an optimized workflow for transcriptomic profiling from PFA-fixed archival retina. This refined methodology paves the way for improved transcriptomic analysis of preserved ocular tissue, bridging the gap between optimal sample preservation and high-quality RNA data acquisition.

P-069 The expression patterns of ALOX15 and GPX4 in the seminiferous epithelium of men with non-obstructive azoospermia (NOA) due to primary testicular failure

Abstract Study question Does the expression of ALOX15 and GPX4 differ in NOA men with a history of cryptozoospermia from NOA who never had sperm in their ejaculate? Summary answer There was no significant difference between these 2 groups of patients. What is known already Oxidative stress (OS) has been investigated as a prime suspect for many unexplained pathologies, including primary testicular failure (PTF). The fact that male germ cells are rich in polyunsaturated fatty acids (PUFAs) but have a limited amount of cytoplasmic scavenging enzymes, is the reason behind their extreme vulnerability to OS. The accumulation of lipid peroxidation markers leads to the inactivation of glutathione peroxidase 4 (GPX4), the lipid repair enzyme, and the exacerbation of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that promotes lipid degradation. This study investigates the activity of these antagonizing enzymes in the testicular tissue of NOA patients. Study design, size, duration This observational study was conducted in the period from May 2020, following the Ethics Committee of the University of Split School of Medicine approval, to October 2023. The testicular tissue from 57 patients diagnosed with NOA (according to WHO criteria) and referred to their first microTESE as a part of fertility treatment was collected and processed. Participants/materials, setting, methods Samples were prepared for histological evaluation and immunodetection (fixation; embedding in paraffin; H&E staining; and staining with anti-ALOX15 and anti-GPX4 antibodies). Two groups of NOA patients were observed: Crypto-NOA and NOA group. The main outcome measure was the number of ALOX15 positive germ cells per 1 mm² of the seminiferous tubule surface (N/ mm²) and the percentage (%) of GPX4 positive surface per surface of the tubule measured by the ImageJ program. Main results and the role of chance Spermatozoa were retrieved in 37 of 57 (64,91 %) men; samples of 4 patients had to be excluded due to poor fixation. There were 18 patients in the Crypto-NOA group, and 10 in the NOA group, while 5 patients were diagnosed with obstructive azoospermia (OA) and were used as controls. For comparison of the baseline data of Crypto-NOA and NOA patients, we used the Mann-Whitney test, and with statistical significance set at the level of P < 0.05, we found that there were significantly more patients with cryptorchism in the NOA group. As a tool for histological evaluation, we used the diagnostic code designed by McLachlan et al. and found similar spermatogenic phenotypes between Crypto NOA and NOA patients. The most represented phenotype was hypospermatogenesis with a moderate reduction in late spermatid number mixed with normal tubules (38,8 % in Crypto-NOA; 40% in NOA), following the phenotype where late spermatids are present in some tubules but mixed with tubules showing no progression past the spermatocyte stage. Using the Mann-Whitney test, we did not find a significant difference in the expression of ALOX15 and GPX4 in the seminiferous epithelium of Crypto-NOA and NOA patients. Limitations, reasons for caution The testicular tissue of NOA patients is characterized by focal or patchy spermatogenesis which led to the exclusion of several samples due to the fixation of tissue with a homogenous pattern of Sertoli cell-only or germ-cell arrest. Wider implications of the findings This study will provide additional information regarding the cause of impaired spermatogenesis and help to determine if the patient is a potential candidate for antioxidant therapy. Our next step is to associate the expression patterns of ALOX15 and GPX4 with the success rates of in vitro fertilization procedure outcomes. Trial registration number not applicable

Assessment of formalin preserved and fresh frozen quadriceps tendon graft-suture constructs for load to failure testing: a biomechanical cadaveric study.

Fresh-frozen specimen availability and cost may be a barrier for initiation of biomechanical studies where soft tissue is used in a construct with other medical devices. The impact of soft tissue preservation method on the outcomes of biomechanical studies in the specific case of graft-suture constructs is relatively unexplored. This study aimed to observe peak loads and failure modes in biomechanical testing of fresh-frozen (FF) versus formalin embalmed (FE) quadriceps tendon (QT) graft-suture constructs for soft tissue fixation in ACLR and assess suitability of FE QT graft constructs for load-to-fail testing. Twenty QT grafts were harvested from human cadaver specimens. Ten grafts came from fresh-frozen donors and 10 from embalmed donors. All grafts were prepared with the modified Prusik knot using a braided composite suture and subjected to tensile loading. Comparisons between the biomechanical properties of the graft-suture constructs were made with unpaired t tests with α = 0.05. FE and FF constructs displayed similar peak loads and failure modes. FF constructs had greater elongation after pre-tensioning than FE (7.3 vs. 5.5mm, p = 0.02) and greater elongation after cyclic loading than FE constructs (17.5 vs. 10.5mm, p = 0.01). Hysteresis was greater for FF constructs at the 50th, 100th, 150th, and 200th cycle (p = 0.02, p = 0.07, p < 0.001, p = 0.004, respectively). FE constructs were stiffer than fresh-frozen (103 vs. 84N/mm, p < 0.001). FE constructs were significantly stiffer but displayed similar peak load and failure mode to FF which was reflective of the strength of the suture material. FE grafts can offer an alternative to FF grafts in graft-suture constructs for biomechanical studies where load at failure and knot security and strength is of main interest.

A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Studi Morfologi Kelenjar Aksesoris Kelamin Jantan Sapi Sumba Ongole (Bos indicus)

Sumba Ongole cattle are one of the local types of cattle that are most commonly found in Indonesia. Bulls have accessory gland are ampulla, vesicular glands, prostate glands, and bulbourethral glands. The aim of this research is to determine the anatomical morphology and histology of male accessory glands in Sumba Ongole cattle. The research sample was six Sumba Ongole cattle that had been slaughtered at the East Sumba Regency Slaughterhouse. Macroscopic examination carried out tissue fixation in 10% formalin, tissue processing, and hematoxylin-eosin staining. The results of the research show that the male accessory glands in Sumba Ongole cattle have the ampullary gland, vesicular gland, prostate gland and bulbourethral gland.. Microscopically, each gland is covered by a capsule and has a secretory gland. The ampullary secretory glands are lined by cylindrical epithelium and have a serous type. The secretory glands of the vesicular glands are lined with cylindrical epithelium and have a seromucous type, the secretory glands of the prostate gland are lined with cuboidal epithelium and have a seromucous type, and the secretory glands of the bulbourethral glands are lined by cuboidal epithelium and have the mucous type.