Tissue Fixation Research Articles

Fixation and staining methods for macroscopical investigation of the brain.

The proper preservation of human brain tissue is an indispensable requirement for post-mortem investigations. Neuroanatomical teaching, neuropathological examination, neurosurgical training, basic and clinical neuroscientific research are some of the possible downstream applications of brain specimens and, although much apart from one another, proper tissue fixation and preservation is a common denominator to all of them. In this review, the most relevant procedures to fixate brain tissue are described. In situ and immersion fixation approaches have been so far the most widespread ways to deliver the fixatives inside the skull. Although most of them rely on the use of formalin, alternative fixative solutions containing lower amounts of this compound mixed with other preservative agents, have been attempted. The combination of fixation and freezing paved the way for fiber dissection, particularly relevant for the neurosurgical practice and clinical neuroscience. Moreover, special techniques have been developed in neuropathology to tackle extraordinary problems, such as the examination of highly infective specimens, as in the case of the Creutzfeldt-Jakob encephalopathy, or fetal brains. Fixation is a fundamental prerequisite for further staining of brain specimens. Although several staining techniques have been developed for the microscopical investigation of the central nervous system, numerous approaches are also available for staining macroscopic brain specimens. They are mostly relevant for neuroanatomical and neuropathological teaching and can be divided in white and gray matter staining techniques. Altogether, brain fixation and staining techniques are rooted in the origins of neuroscience and continue to arouse interest in both preclinical and clinical neuroscientists also nowadays.

A simple, flexible, and high-efficiency western blot analysis for age-related human induced neurons.

High-throughput western blot (WB) analysis can be used to obtain more consistent, comparable, and informative data from precious samples and materials with extremely limited availability, such as various age-related, subtype-specific human induced neurons (hiNs). In this study, p-toluenesulfonic acid (PTSA), an odorless tissue fixative, was used to inactivate horseradish peroxidase (HRP) and develop a high-throughput WB method. PTSA-treated blots demonstrated rapid and efficient HRP inactivation without detectable protein loss or epitope damage. With a brief PTSA treatment (1min at room temperature [RT]) before every subsequent probing, 10 dopaminergic hiN proteins could be sequentially, sensitively, and specifically detected in the blot. The resulting WB data confirmed the age-associated and neuron-specific features of hiNs and revealed a significant reduction in two Parkinson's disease-associated proteins, UCHL1 and GAP43, in normal aging dopaminergic neurons. Overall, this study developed a unique and high-efficiency WB analysis method for capturing robust and useful data from limited, precious samples.

Impact of overfixation on actionable biomarkers and checkpoint inhibitor (CKI) targets with immunohistochemistry (IHC) in a patient population with non-small cell lung cancer (NSCLC).

e15144 Background: The treatment of patients with advanced NSCLC has become reliant on tissue specimens and biomarkers to help guide appropriate treatment options. Currently, there are numerous lung cancer biomarker-defined patient subgroups, with evidence showing that fixation conditions may alter their expression on FFPE specimens, but little evidence on the ensemble of targetable biomarkers and CKI targets. Methods: We assessed 30 adult patients with primary NSCLC tumors. 25 specimens were properly fixed with neutral 10% formalin using gentle agitation for 24-48 hours, whereas 5 specimens were over-fixed (52-108 hours). We examined expression of actionable and exploratory biomarkers ALK, ROS1, BRAF, EGFR, c-Met, panTRK, HER2 and MEK1. We also examined CKI targets such as PDL1, CD3/CD8, CD3/CD8/FoxP3, PD1, CD3/CD8/PD1. We performed IHC on all slides which were scored by a thoracic pathologist per standard clinical practice. Descriptive statistics were applied to further explore the impact of overfixation on IHC results. Results: Results are shown in the table below. Patients had an average age of 64, were mostly male (22/30) with adenocarcinoma histology (21/30). Specimens were collected in compliance with local rules and regulations and tumor staging was from IB-IV. For the 5 overfixed specimens, multiplex PD-L1 was less expressed when compared to the normally fixed specimens. We also surprisingly observed that cMet and HER2 had stronger expression with overfixed tissue. All other targets had no differences observed in this small patient population. Conclusions: Overfixation of NSCLC FFPE had little to no detrimental impact on most targetable biomarkers, except multiplex PD-L1. We also observed that cMet and HER2 had a slightly stronger expression level with overfixed tissue. As we do not have a reference of the overfixed samples fixed from 24-48 hrs, we cannot be assured that the differences in expression levels are not inherent to the sample itself and independent of fixation time. Additional tests of the same sample fixed over different periods will help to identify the true cause. We propose a consistent approach on tissue fixation time and process as the ensemble of preanalytical conditions is critical in ensuring proper biomarker characterization and validation with IHC. [Table: see text]

Myelin histology: a key tool in nervous system research.

The myelin sheath is a lipoprotein-rich, multilayered structure capable of increasing conduction velocity in central and peripheral myelinated nerve fibers. Due to the complex structure and composition of myelin, various histological techniques have been developed over the centuries to evaluate myelin under normal, pathological or experimental conditions. Today, methods to assess myelin integrity or content are key tools in both clinical diagnosis and neuroscience research. In this review, we provide an updated summary of the composition and structure of the myelin sheath and discuss some histological procedures, from tissue fixation and processing techniques to the most used and practical myelin histological staining methods. Considering the lipoprotein nature of myelin, the main features and technical details of the different available methods that can be used to evaluate the lipid or protein components of myelin are described, as well as the precise ultrastructural techniques.

Evaluation of the influence of the formalin fixation time on the elemental content of tissues measured with X-ray fluorescence

One of the most widely used methods for tissue preservation and fixative during transportation is the conservation in formalin during variable amounts of time. In this study, we have evaluated the influence of formalin fixation time in the elemental composition of human tissue samples using Energy Dispersive X-Ray Fluorescence (EDXRF).Ten sets of human tissue samples (from colon, ileum, stomach, and spleen) were exposed to different formalin fixation times, between 2 and 24 days, and for each tissue, the elemental content throughout time was compared to the elemental content of the snap-frozen sample of the same tissue that was not exposed to formalin. Additionally, in order to further evaluate the transference of elements, the formalin solution was also analysed using EDXRF.Our results showed a clear decrease of Cl and K in the tissues, transferred to the formalin solution. Conversely, there is an uptake of P in the tissue, likely due to the buffered formalin solution. The consistent alterations seen in the studied elements across all the ten different tissues allow us to hypothesize that in the future, there will be different thresholds for their use as diagnostic tools in unfixed (intraoperative exams) as well as formalin fixed paraffin embedded tissues.

Epitope Lability of Phosphorylated Biomarkers of the DNA Damage Response Pathway Results in Increased Vulnerability to Effects of Delayed or Incomplete Formalin Fixation.

Phosphorylated biomarkers are crucial for our understanding of drug mechanism of action and dose selection during clinical trials, particularly for drugs that target protein kinases, such as DNA-damage-response (DDR) inhibitors. However, tissue fixation conditions needed to preserve DDR-specific phospho-biomarkers have not been previously investigated. Using xenograft tissues and tightly controlled formalin fixation conditions, we assessed how preanalytical factors affect phosphorylated DDR biomarkers pRAD50(Ser635), ɣH2AX(Ser139), pKAP1(Ser824), and non-phosphorylated biomarkers cMYC and ATM. Cold ischemia times ranged from 15 min to 6 hr, and the fixation duration ranged from 24 hr to 4 weeks. Epitopes pRAD50 and pKAP1 appeared the most labile assessed with staining loss after just 15 min of cold ischemia time, while ATM was more robust showing consistent expression up to 1 hr of cold ischemia. Notably, ɣH2AX expression was lost with formalin fixation over 48 hr. The use of core needle biopsies where possible and novel fixation methods such as the 2-step temperature-controlled formalin approach may improve phosphorylated biomarker preservation; however, practical challenges may affect wider clinical application. The most essential tissue-processing step when downstream analysis includes DDR phosphorylated biomarkers is immediate tissue submersion in formalin, without delay, upon excision from the patient, followed by room temperature fixation for 24 hr.

FixNCut: Single-cell genomics through reversible tissue fixation and dissociation

FixNCut: Single-cell genomics through reversible tissue fixation and dissociation

Formalin-free fixation and xylene-free tissue processing preserves cell-hydrogel interactions for histological evaluation of 3D calcium alginate tissue engineered constructs

Histological evaluation of tissue-engineered products, including hydrogels for cellular encapsulation, is a critical and invaluable tool for assessing the product across multiple stages of its lifecycle from manufacture to implantation. However, many tissue-engineered products are comprised of polymers and hydrogels which are not optimized for use with conventional methods of tissue fixation and histological processing. Routine histology utilizes a combination of chemical fixatives, such as formaldehyde, and solvents such as xylene which have been optimized for use with native biological tissues due to their high protein and lipid content. Previous work has highlighted the challenges associated with processing hydrogels for routine histology due to their high water content and lack of diverse chemical moieties amenable for tissue fixation with traditional fixatives. Thus, hydrogel-based tissue engineering products are prone to histological artifacts during their validation which can lead to challenges in correctly interpreting results. In addition, chemicals used in conventional histological approaches are associated with significant health and environmental concerns due to their toxicity and there is thus an urgent need to identify suitable replacements. Here we use a multifactorial design of experiments approach to identify processing parameters capable of preserving cell-biomaterial interactions in a prototypical hydrogel system: ionically crosslinked calcium alginate. We identify a formalin free fixative which better retains cell-biomaterial interactions and calcium alginate hydrogel integrity as compared to the state-of-the-art formalin-based approaches. In addition, we demonstrate that this approach is compatible with a diversity of manufacturing techniques used to fabricate calcium alginate-based scaffolds for tissue engineering and cell therapy, including histological evaluation of cellular encapsulation in 3D tubes and thin tissue engineering scaffolds (∼50 μm). Furthermore, we show that formalin-free fixation can be used to retain cell-biomaterial interactions and hydrogel architecture in hybrid alginate-gelatin based scaffolds for use with histology and scanning electron microscopy. Taken together, these findings are a significant step forward towards improving histological evaluation of ionically crosslinked calcium alginate hydrogels and help make their validation less toxic, thus more environmentally friendly and sustainable.

Using Antibodies in Microscopy: A Guide to Immunohistochemistry. Part 1: Antibodies, Tissue Fixation, Embedding, and Sectioning

AbstractImmunohistochemistry (IHC) is the use of antibodies as probes to determine the localization of proteins in a cell or tissue sample. In microscopy, the technique has been in use since the 1940s by pathologists and research scientists. At first glance it appears to be a straightforward procedure. In practice, however, an IHC protocol has several steps where problems can occur, resulting in either false positive or false negative data. IHC can be used in both transmitted light and fluorescence microscopy, and it can be used in combinations to identify and mark multiple proteins of interest. While many IHC techniques have been published, a certain amount of confusion exists as to how to optimize IHC procedures. This series of three articles will describe the steps involved in IHC, the function of each step, and some variations that occur in specific protocols on differing cell and tissue types. It is hoped that a better understanding of these steps will help to guide users in determining the optimal conditions for carrying through IHC protocols.

Effects of Bipolar Radiofrequency on Collagen Synthesis from Patients with Brachial Ptosis.

Inclusion criteria included patients with stage 1, 2a, and 2b brachial ptosis (Duncan classification) and upper limb fat deposits who were considered candidates for third-generation ultrasound-assisted liposculpture plus radiofrequency-assisted lipolysis/skin tightening. Arm subdermal tissue samples (5 mm³) were analyzed before and after the intervention. We used 10% formaldehyde for tissue fixation and stained each sample with hematoxylin/eosin, Masson trichrome, and antibody markers against the cell cycle Ki-67 protein. We analyzed a total of 12 biopsies from six patients who meet the inclusion/exclusion criteria. Histological findings with hematoxylin/eosin revealed hyperplastic and metaplastic changes with focal distribution within the papillary and reticular dermis. Masson trichrome staining showed an increase of the characteristic basophilia of thin type-I and type-III collagen fibers. In contrast, molecular analysis reported an increase in fibroblast activity mediated by the activation of the heat shock protein HSP47. Radiofrequency may be a great alternative to improve skin retraction in patients with mild to moderate brachial dermatochalasis through the activation of HSP47 heat shock protein and the production of type-I and type-III collagen.

Machine learning classification of placental villous infarction, perivillous fibrin deposition, and intervillous thrombus

IntroductionPlacental parenchymal lesions are commonly encountered and carry significant clinical associations. However, they are frequently missed or misclassified by general practice pathologists. Interpretation of pathology slides has emerged as one of the most successful applications of machine learning (ML) in medicine with applications ranging from cancer detection and prognostication to transplant medicine. The goal of this study was to use a whole-slide learning model to identify and classify placental parenchymal lesions including villous infarctions, intervillous thrombi (IVT), and perivillous fibrin deposition (PVFD). MethodsWe generated whole slide images from placental discs examined at our institution with infarct, IVT, PVFD, or no macroscopic lesion. Slides were analyzed as a set of overlapping patches. We extracted feature vectors from each patch using a pretrained convolutional neural network (EfficientNetV2L). We trained a model to assign attention to each vector and used the attentions as weights to produce a pooled feature vector. The pooled vector was classified as normal or 1 of 3 lesions using a fully connected network. Patch attention was plotted to highlight informative areas of the slide. ResultsOverall balanced accuracy in a test set of held-out slides was 0.86 with receiver-operator characteristic areas under the curve of 0.917–0.993. Cases of PVFD were frequently miscalled as normal or infarcts, the latter possibly due to the perivillous fibrin found at the periphery of infarctions. We used attention maps to further understand some errors, including one most likely due to poor tissue fixation and processing. DiscussionWe used a whole-slide learning paradigm to train models to recognize three of the most common placental parenchymal lesions. We used attention maps to gain insight into model function, which differed from intuitive explanations.

Histology of the whole body of honey bees: tissue fixation and processing.

Beekeeping plays a crucial role in biodiversity, pollination, commercial farming, and the worldwide agricultural economy. Histopathology, which is an important tool for the investigation of diseases in vertebrates, is not commonly used in honey bees (Apis mellifera). However, histopathology could potentially help the diagnostic investigation of high mortality in bees. We developed a tissue fixation and processing method enabling systematic production of histologic slides adequate for diagnostic and research purposes. Our method uses inexpensive, accessible products and can be realized with conventional pathology laboratory equipment. The quality of histologic slides obtained is similar to those of vertebrate animals processed routinely in pathology laboratories. Histopathology as a diagnostic and research tool will improve the services currently offered to apiarists and could help decrease the mean mortality rate, increase apiarists' profits, and ensure long-term pollination services.

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

ObjectivesThe objective is to assess the impact of the quality of tissue fixation in surgical pathology on immunohistochemical (IHC) staining and DNA degradation. Materials and methodsTwenty-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. ResultsH-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). ConclusionsImpaired 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.

The Application of Guanidinium to Improve Biomolecule Quality in Fixed, Paraffin-embedded Tissue.

Neutral buffered formalin (NBF) is the most common fixative in clinical applications. However, NBF damages proteins and nucleic acids, limiting the quality of proteomic and nucleic acid-based assays. Prior studies have demonstrated that BE70, a fixative of buffered 70% ethanol, has many benefits over NBF but the degradation of proteins and nucleic acids in archival paraffin blocks remain a challenge. Thus, we evaluated the addition of guanidinium salts to BE70 with the hypothesis that this may protect RNA and protein. Guanidinium salt supplemented BE70 (BE70G)-fixed tissue is comparable with that of BE70 via histology and immunohistochemistry. Western blot analysis also revealed that HSP70, AKT, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression signals in BE70G-fixed tissue were higher than those in BE70-fixed tissue. The quality of nucleic acids extracted from BE70G-fixed, paraffin-embedded tissue was also superior, and BE70G provides improved protein and RNA quality at shorter fixation times than its predecessors. The degradation of proteins, AKT and GAPDH, in archival tissue blocks is also decreased with the addition of guanidinium salt to BE70. In conclusion, BE70G fixative improves the quality of molecular analysis with more rapid fixation of tissue and enhanced long-term storage of paraffin blocks at room temperature for evaluation of protein epitopes.

Mid-Term Clinical and Radiological Outcomes of Lateral Meniscal Allograft Transplantation with Suture-Only Fixation Plus Capsulodesis.

Meniscal allograft transplantation (MAT) is an effective reconstructive procedure for treating a symptomatic postmeniscectomy syndrome. It consists of replacing the lost meniscal tissue aiming to improve the clinical outcomes and prevent progressive deterioration of the joint. The aim of this study was to evaluate meniscal graft survivorship and report on the radiographic (in terms of graft extrusion and joint space width and alignment) and the functional results through a midterm follow-up of lateral MAT performed with a soft tissue fixation technique after capsulodesis. In total, 23 patients who underwent lateral MAT as a single procedure were included. The Knee injury and Osteoarthritis Outcome Score, Lysholm, Tegner, and visual analog scale scales were used for patient assessment. Magnetic resonance imaging and a complete radiographic protocol were conducted to determine the degree of meniscal extrusion and the changes in the degree of osteoarthritis and coronal alignment. Assessments were performed after 2 and 7 years of follow-up. A significant improvement in all the scores, relative to preoperative values, was found after 7 years of follow-up. This improvement remained consistent throughout the first and second follow-up periods. A mean absolute extrusion of 2.2 mm ± 1.6 and an extrusion percentage of 28.0% ± 11.43 were found, with no significant differences throughout the follow-up periods. There was no statistically significant difference in terms of the frontal mechanical axis and joint space narrowing between the preoperative value and at the first and second follow-up periods. A survival rate of 85.7% was found after 7 years of follow-up. Capsulodesis results in a low degree of meniscal extrusion in isolated lateral MAT fixed with a suture-only technique, which is maintained after 7 years of follow-up, with a high graft survival index (>85%) and satisfactory results on the functional scales.

Elderly with Varying Extents of Cardiac Disease Show Interindividual Fluctuating Myocardial TRPC6-Immunoreactivity

Both particular myocardial locations in the human heart and the canonical transient receptor potential 6 (TRPC6) cation channel have been linked with cardiac pathophysiologies. Thus, the present study mapped TRPC6-protein distribution in select anatomic locations associated with cardiac disease in the context of an orienting pathological assessment. Specimens were obtained from 5 body donors (4 formalin fixation, 1 nitrite pickling salt-ethanol-polyethylene glycol (NEP) fixation; median age 81 years; 2 females) and procured for basic histological stains and TRPC6-immunohistochemistry. The latter was analyzed descriptively regarding distribution and intensity of positive signals. The percentage of positively labelled myocardium was also determined (optical threshold method). Exclusively exploratory statistical analyses were performed. TRPC6-protein was distributed widespread and homogenously within each analyzed sample. TRPC6-immunoreactive myocardial area was comparable regarding the different anatomic regions and sex. A significantly larger area of TRPC6-immunoreactive myocardium was found in the NEP-fixed donor compared to the formalin fixed donors. Two donors with more severe heart disease showed smaller areas of myocardial TRPC6-immunoreactivity overall compared to the other 3 donors. In summary, in the elderly, TRPC6-protein is widely and homogenously distributed, and severe cardiac disease might be associated with less TRPC6-immunoreactive myocardial area. The tissue fixation method represents a potential confounder.

Qualitative assessment of Aloe vera as natural tissue fixative: An institutional study.

The art of microscopy is only appreciable in well-fixed tissues. We conducted this study to determine the effectiveness of Aloe vera as a tissue fixative and compare its results with the natural fixatives already studied in the literature. A pilot study was tried out using commercially available fresh chicken and fish with Aloe vera, and then after getting the promising results similar study protocol was carried out using 10-autopsied human tissue. Four natural fixatives-30% jaggery solution, 20% honey solution, 20% sugar solution, 20% Aloe vera solution and 10% formalin were used for fixation in the study. Fixation of tissues was carried out at room temperature for 24 h. All pre- and postfixation measurements were recorded using stereomicroscope and its software. The difference between pre-and postfixation was calculated and later, all pieces were kept for routine tissue processing followed by routine staining. The tissue sections were assessed for quality, and the whole procedure was blinded among three oral pathologists who scored them. The mean percentage of shrinkage in each bit with different reagents was calculated. The shrinkage seen with 10% formalin and 20% Aloe vera were more likely similar. Among all the natural fixatives, qualitatively also Aloe vera excelled and its results were comparable to that of formalin. The use of Aloe vera in the present study as fixative is the first of its kind, as exhaustive search of the literature shows only its use as transport media in dentistry.

Dissecting Collective Cell Behavior in Migrating Testis Myotubes in Drosophila.

Collective cell migration has a key role in tissue morphogenesis, wound healing, tissue regeneration, and cancer invasion. In recent years, different animal models have been established to analyze how chemical and mechanical stimuli shape the behavior of single cells into tissues and organs. At present, there are still only a few model systems that allow to genetically dissect underlying molecular mechanisms driving cell motility during tissue morphogenesis at high resolution in real time. Here, we provide a detailed protocol and toolbox for ex vivo culturing of Drosophila testes for 4D live imaging of myotube collective migration, which allows to genetically address a wide range of developmental and cell biological questions regarding modes of filopodia-based protrusion/locomotion, cell-cell adhesion, cytoskeletal modes of collective decision-making, and collective closure processes. Additionally, this protocol has been successfully used in combination with laser-induced single-cell ablation and pharmacological treatments, but it can also be used with confocal microscopy after tissue fixation.

A prospective study to evaluate the functional and clinical outcome of anterior cruciate ligament reconstruction with standard hamstring autograft

Background: The anterior cruciate ligament (ACL) is the primary stabilizer of the knee and prevents the knee against anterior translation. It is also important in counteracting rotational and valgus stress. The arthroscopically aided approach has the advantages of smaller skin and capsular incisions, improved viewing of the intercondylar notch for placement of the tunnel and attachment sites, less postoperative pain, fewer adhesions, earlier motion, and easier rehabilitation. The advantages with semitendinosus grafts are the availability of a greater sectional area and the unaffected integrity of the extensor mechanism. Moreover, the tensile strength of quadrupled hamstring is found to be even better than a normal ACL. The rate of complications such as extension loss, anterior knee pain, and surgical site infections was also lower than in other procedures such as in BPTB grafts. Cortical suspension devices have the necessary biomechanical properties with regard to ultimate failure strength, displacement, and stiffness for the initial fixation of soft tissue in the femoral tunnel for ACL reconstruction. The aperture fixation methods such as the interference screws allow for early firm fixation and heal with a tight bone–tendon interface.Objective: A prospective study to evaluate the functional and clinical outcome of ACL reconstruction with standard hamstring autograft.Materials and Methods: The study results included 30 patients admitted with clinical and radiological deficiency of ACL from September 2019 to March 2021 using endobutton and bioabsorbable interference screw in terms of range of motion, postoperative knee stability, subjective knee functions, and prospective assessment of functional outcome using Tegner Lysholm knee scoring system.Results: Sports injuries were the most common mode of injury (n = 17, 56.7%). The mean size of graft used was 8.2 ± 0.39 mm. Postoperatively at 2-year follow-up, 30 cases (100%) had 0 grade pivot-shift test, 0 grade Lachman test, and anterior drawer test. The mean postoperative Lysholm score improved from preoperative score of 62.83 ± 5.2–91.0 ± 5.54. The overall satisfactory result (excellent + good) at 6-month follow-up was (excellent – 30 and good – 70) 100%. Out of this, 27 (90%) patients had returned to preinjury level of activity in 1 year. In this study, one patient had superficial infection and two patients had knee pain.Conclusion: Arthroscopy-assisted ACL reconstruction using hamstring tendon autograft provides a stable knee, reduces postoperative morbidity, and enables early rehabilitation.

Optimization of Testicular Fixation-Embedding Techniques for Improved Evaluation of Mammalian Spermatogonial Morphology and Function.

Numerous methods have beensuccessfully used to evaluate mammalian spermatogonial biologyHowever, the conventional light microscopy assays present a challenge in precisely identifying spermatogonial phenotypes, which can result in discrepancies between molecular and morphological findings. Such precise association could lead to a more robust interpretation of spermatogonial activityin steady-state spermatogenesis, which may facilitate the translation from basic researchto clinical applications. In this chapter, we present two histological processing methods that enable a comprehensive analysis of spermatogonial morphology and function, involving fixation of mammaliantesticular tissue in glutaraldehyde and embedding in plastic resin. These techniques have proven to be effective in light microscopy studies.